Anaplastic Large Cell Lymphoma Patients Research Articles

Abstract 1113: Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models

Abstract Neoplastic cells are characterized by genetic instability, leading to the random accumulation of mutations in different tumor subpopulations, which therefore respond differently to treatments. Given the average mutational rate of tumors, the probability to acquire resistance to a single drug usually exceeds the number of cells present at diagnosis, making drug resistance a fact in most cases. Statistically, the chance of a cell being resistant to two simultaneous treatments is much lower, suggesting that drug combinations may help prevent resistance. Anaplastic large cell lymphoma (ALCL) is an aggressive type of Non-Hodgkin Lymphoma, characterized by expression of the NPM/ALK oncogenic fusion tyrosine kinase. ALK+ ALCL patients resistant or relapsed (R/R) to front-line chemotherapy have a very poor prognosis. The therapeutic activity of the ALK inhibitor crizotinib has been shown in patients with R/R ALK+ ALCL. However, 30-40% of patients develop resistance to crizotinib monotherapy and relapse within short time, leaving no further therapeutic option than bone marrow transplant, when feasible. This highlights the need to identify new first-line therapies for high-risk patients.We explored the potential of upfront drug combinations to prevent the rise of resistant disease, in vitro and in vivo. Crizotinib was combined with CHOP chemotherapy, an epigenetic drug (decitabine), another targeted inhibitor (trametinib- MEK inhibitor), or a second-generation ALK inhibitor. ALK+ ALCL cells were kept in culture for ~100 days in the presence of different single or combined drugs. We found that in most cases, combined treatments were more effective than single agents in the long-term control of cells expansion. Upon further functional characterization using quantitative PCR and western blot, we found that combination treatments upregulated cell cycle inhibitors and pro-apoptotic genes as well as modulated survival factors to induce deeper inhibition of oncogenic signalling and higher rates of apoptosis. Crizotinib+decitabine combination treatment promoted downregulation of DNMT1 and increase of TGFB1, both of which indirectly affect cell cycle progression. In murine model, crizotinib+decitabine significantly delayed tumour growth, compared to single treatments. Our results indicate that combining a selective drug with a less specific anticancer agent, which allows unspecific killing of resistant clones that might potentially arise, would be beneficial.In conclusion, our data suggest that a polypharmacology approach combining ALK inhibition with other agents could be a valuable therapeutic option for ALK+ ALCL patients, to prevent relapses and improve outcomes. Citation Format: Geeta G. Sharma, Giulia Arosio, Matteo Villa, Mario Mauri, Marina Zappa, Vera Magistroni, Monica Ceccon, Sara Redaelli, Ilaria Crespiatico, Diletta Fontana, Cristina Mastini, Anna Garbin, Federica Lovisa, Lara Mussolin, Rocco Piazza, Carlo Gambacorrti-Passerini, Luca Mologni. Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1113.

Evaluation of the NCCN-IPI As a Prognostic Tool in PTCLs

Background: The majority of systemic peripheral T-cell lymphomas (PTCLs) have an aggressive behavior and compared to diffuse large B-cell lymphoma, survival is inferior using standard anthracycline-based treatment. Patient and disease factors impact survival, and the International Prognostic Index (IPI) is the most widely used clinical tool for prognostication for aggressive lymphomas. The IPI has shown prognostic utility in some PTCL entities such as systemic anaplastic large cell lymphoma (ALCL) and PTCL not otherwise specified (PTCL NOS), but less usefulness for others such as angioimmunoblastic T-cell lymphoma (AITL) as well as the rarer predominantly extranodal PTCL subtypes. T-cell specific prognostic indices such as the Prognostic Index for PTCL-U (PIT) have been explored but the IPI remains the most commonly used prognostic tool. The NCCN-IPI prognostic index has recently been developed for DLBCL. The NCCN-IPI builds on much the same parameters as the IPI, but with age stratified into four groups, lactate dehydrogenase (LDH) ratio using the upper limit of normal (ULN) separated into three categories (<1 ULN, 1-3 ULN, >3 ULN) and it also incorporates specific extranodal sites (CNS, gastrointestinal, lung or bone marrow involvement) but not absolute number of sites, with a maximum score of 8 points. In this study we evaluated the NCCN-IPI in comparison to the IPI and PIT in the three main nodal PTCL subgroups: ALK-neg ALCL, AITL, and PTCL NOS.Methods: A cohort of newly diagnosed adult PTCL patients treated with anthracyline-based or other curative intent chemotherapy regimens was assembled from the Swedish Lymphoma Registry (SWE), University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER), and British Columbia Cancer Agency (BCCA). Patients with complete prognostic data and ALK-neg ALCL, AITL or PTCL NOS subtypes were included. Event-free survival (EFS) was defined as time from pathologic diagnosis to progression/relapse, retreatment, or death due to any cause. Kaplan-Meier curves and Cox proportional hazards models were used to examine prognostic utility of indices; concordance was measured via Harrell's c-index.Results: In total, 603 patients were included. The majority of AITL presented with advanced stage disease (92%) and elevated LDH (75%) while ALK-neg ALCL patients less often exhibited adverse factors (advanced disease [67%] and elevated LDH [50%]) and PTCL NOS patients showed characteristics in between. At a median follow-up of 81 months (range 1-185), 449 patients (75%) had an event and 400 patients (66%) had died. Kaplan-Meier curves for EFS by NCCN-IPI are shown in figure 1. In patients with AITL (N=145), the NCCN-IPI had limited prognostic utility (EFS c-index=0.580) which was worse than the IPI (c-index=0.597) and similar to the PIT (c=0.585). The number of AITL patients with low-risk disease by NCCN-IPI was very small (N=2 [1%]). The NCCN-IPI had excellent prognostic utility in N=152 patients with ALK-neg ALCL (c-index=0.683), which was superior to the IPI and PIT (both c-index=0.665). However, a limited number of patients were classified as low risk (15%) or high risk disease (8%). The NCCN-IPI had modest prognostic utility in 306 patients with PTCL, NOS (c-index=0.629), which was inferior to the IPI (c-index=0.645) and similar to the PIT (c-index=0.631). Similar to ALK-neg ALCL, a limited number of patients were classified as low risk (12%) or high risk (14%) disease.Conclusions: The NCCN-IPI performed similarly to the IPI with the exception of ALK-neg ALCL where it appeared to be better at defining low and very high risk patients. In AITL, the NCCN-IPI had very limited prognostic ability and performed even worse than the IPI (which also had limited prognostic ability). Better risk stratification is needed in this disease. For PTCL-NOS, the NCCN-IPI performed similarly to the IPI and PIT. While regrouping of scores could potentially improve this situation, the integration of other clinically defined prognostic factors and molecular characteristics is needed for more precise prognostic models in PTCLs. [Display omitted] DisclosuresEllin:CTI: Consultancy; ROCHE: Consultancy, Research Funding. Connors:NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Research Funding; Seattle Genetics: Research Funding; NanoString Technologies: Research Funding; Lilly: Research Funding; Merck: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Takeda: Research Funding; F Hoffmann-La Roche: Research Funding; Bayer Healthcare: Research Funding. Smedby:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Affimed: Research Funding. Slack:Seattle Genetics: Consultancy. Cerhan:Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering . Savage:Celgene: Consultancy; Roche: Research Funding; Seattle Genetics: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Merck: Honoraria.

Aberrant expression and genetic alteration of c-MYC in anaplastic large cell lymphoma.

c-MYC plays an important role in regulating cellular growth and apoptosis, and it is aberrantly expressed in many human malignancies. Although c-MYC has been extensively investigated in Burkitt lymphoma and diffuse large B cell lymphoma, little has been reported in anaplastic large cell lymphoma (ALCL). The aim of this study was to investigate the expression and genetic alterations of c-MYC in primary systemic ALCL, characterize its clinicopathologic features and immunophenotypes, and discuss their implications in prognosis. Tissue microarrays using samples from 85 ALCL patients were used to evaluate expression of c-MYC and anaplastic lymphoma kinase (ALK). c-MYC and ALK genetic alterations were detected using fluorescence in situ hybridization. The Kaplan-Meier and multivariate Cox regression methods were used for survival analysis. c-MYC was expressed in 24 of 85 samples (28.2%), and ALK was expressed in 54 (63.5%). c-MYC and ALK were co-expressed in 16 samples (18.8%). c-MYC expression and c-MYC and ALK co-expression increased with ALCL clinical stages and the International Prognostic Index (IPI) score (p < 0.05). Fifty of the samples (58.8%) had ALK rearrangement, and 18 (22.1%) had aneuploidy. c-MYC rearrangement was not detected, but aneuploidy was observed in 19 cases (22.4%). c-MYC aneuploidy was significantly different based on c-MYC expression and the IPI score (p < 0.05). c-MYC was a significant independent prognostic factor for progression-free survival and overall survival in patients with ALCL. c-MYC protein expression and c-MYC aneuploidy could predict worse survival in patients with ALCL.

MDMX/MDM4 is highly expressed and contributes to cell growth and survival in anaplastic large cell lymphoma

We hypothesized that murine double minute X (MDMX), a negative p53-regulator, may be involved in dysfunctional p53-signaling in anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK)-positive and ALK-negative, characterized frequently by non-mutated TP53 (wt-p53). By western blot analysis, MDMX was highly expressed in ALK + ALCL and expressed at variable levels in ALK- ALCL cell lines. By immunohistochemistry, high MDMX levels were observed more frequently in ALK + ALCL (36/46; 78%), compared with ALK- ALCL tumors (12/29; 41%) (p < .0018, Mann–Whitney-test). FISH analysis showed MDMX-amplification in 1 of 13 (8%) ALK- ALCL tumors, and low-level MDMX copy gains in 2 of 13 (15%) ALK- ALCL and 3 of 11 (27%) ALK + ALCL tumors. MDMX-pharmacologic inhibition or siRNA-mediated MDMX-silencing were associated with activated p53 signaling, growth inhibition and apoptotic cell death in wt-p53 ALCL cells, providing evidence that targeting MDMX may provide a new therapeutic approach for ALCL patients with wt-p53.

Increased Tenascin C, Osteopontin and HSP90 Levels in Plasmatic Small Extracellular Vesicles of Pediatric ALK-Positive Anaplastic Large Cell Lymphoma: New Prognostic Biomarkers?

Over the past 15 years, several biological and pathological characteristics proved their significance in pediatric anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL) prognostic stratification. However, the identification of new non-invasive disease biomarkers, relying on the most important disease mechanisms, is still necessary. In recent years, plasmatic circulating small extracellular vesicles (S-EVs) gathered great importance both as stable biomarker carriers and active players in tumorigenesis. In the present work, we performed a comprehensive study on the proteomic composition of plasmatic S-EVs of pediatric ALCL patients compared to healthy donors (HDs). By using a mass spectrometry-based proteomics approach, we identified 50 proteins significantly overrepresented in S-EVs of ALCL patients. Gene Ontology enrichment analysis disclosed cellular components and molecular functions connected with S-EV origin and vesicular trafficking, whereas cell adhesion, glycosaminoglycan metabolic process, extracellular matrix organization, collagen fibril organization and acute phase response were the most enriched biological processes. Of importance, consistently with the presence of nucleophosmin (NPM)-ALK fusion protein in ALCL cells, a topological enrichment analysis based on Reactome- and Kyoto Encyclopedia of Genes and Genomes (KEGG)-derived networks highlighted a dramatic increase in proteins of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in ALCL S-EVs, which included heat shock protein 90-kDa isoform alpha 1 (HSP90AA1), osteopontin (SPP1/OPN) and tenascin C (TNC). These results were validated by Western blotting analysis on a panel of ALCL and HD cases. Further research is warranted to better define the role of these S-EV proteins as diagnostic and, possibly, prognostic parameters at diagnosis and for ALCL disease monitoring.

NPM-ALK: A Driver of Lymphoma Pathogenesis and a Therapeutic Target.

Simple SummaryAnaplastic lymphoma kinase (ALK) is a tyrosine kinase associated with Anaplastic Large Cell lymphoma (ALCL) through oncogenic translocations mainly NPM-ALK. Chemotherapy is effective in ALK(+) ALCL patients and induces remission rates of approximately 80%. The remaining patients do not respond to chemotherapy and some patients have drug-resistant relapses. Different classes of ALK tyrosine kinase inhibitors (TKI) are available but used exclusively for EML4-ALK (+) lung cancers. The significant toxicities of most ALK inhibitors explain the delay in their use in pediatric ALCL patients. Some ALCL patients do not respond to the first generation TKI or develop an acquired resistance. Combination therapy with ALK inhibitors in ALCL is the current challenge.Initially discovered in anaplastic large cell lymphoma (ALCL), the ALK anaplastic lymphoma kinase is a tyrosine kinase which is affected in lymphomas by oncogenic translocations, mainly NPM-ALK. To date, chemotherapy remains a viable option in ALCL patients with ALK translocations as it leads to remission rates of approximately 80%. However, the remaining patients do not respond to chemotherapy and some patients have drug-resistant relapses. It is therefore crucial to identify new and better treatment options. Nowadays, different classes of ALK tyrosine kinase inhibitors (TKI) are available and used exclusively for EML4-ALK (+) lung cancers. In fact, the significant toxicities of most ALK inhibitors explain the delay in their use in ALCL patients, who are predominantly children. Moreover, some ALCL patients do not respond to Crizotinib, the first generation TKI, or develop an acquired resistance months following an initial response. Combination therapy with ALK inhibitors in ALCL is the current challenge.

Molecular profiling of Chinese systemic anaplastic large cell lymphoma patients: novel evidence of genetic heterogeneity.

BackgroundAnaplastic large cell lymphoma (ALCL) is a rare non-Hodgkin lymphoma. A comprehensive understanding of the genetic and clinical heterogeneity of ALCL may help to improve the clinical management of patients with ALCL. However, due to the rarity of the disease, the genetic heterogeneity of ALCL has not been well elucidated. This study aimed to comprehensively elucidate the mutational landscape of tumor tissue samples from patients with systemic ALCL.MethodsThirty-six patients with systemic ALCL were enrolled in this retrospective study. Immunohistochemistry (IHC) was performed on tumor tissues at baseline to identify anaplastic lymphoma kinase (ALK) fusions. Capture-based targeted next-generation sequencing (NGS) with a panel spanning 112 lymphoma-related genes, including ALK rearrangements, was also performed on tumor tissue samples.ResultsA total of 102 mutations were identified in the entire cohort. Among the 36 patients included in this analysis, 14 (38.8%) were ALK positive, as determined by IHC, while NGS showed 12 patients (33.3%) to harbor ALK rearrangements. Younger patients were more likely to have ALK-positive ALCL (P=0.011). Patients with wild-type (WT) ALK were more likely to have single-nucleotide variants (SNVs) and insertions or deletions (INDELs) than patients with ALK rearrangements (P=0.027). Among the 22 patients with WT ALK, the most commonly mutated genes were TP53 (n=6, 27.3%), followed by NOTCH1 (n=5, 22.7%), KMT2D (n=3, 13.6%), KRAS (n=3, 13.6%), TET2 (n=3, 13.6%), and JAK1 (n=2, 9.1%). Mutations in PRDM1, a commonly mutated gene in ALK-negative patients, were not detected in our ALK-negative cohort. Start-loss of beta-2-microglobulin (B2M) was detected in another patient; this patient had a favorable prognosis, with an overall survival exceeding 19 months.ConclusionsOur study revealed the unique genomic profiles of Chinese ALCL patients and represents an incremental step in deepening the understanding of the genetic heterogeneity of ALCL patients.

Acute leukemic phase of anaplastic lymphoma kinase-anaplastic large cell lymphoma: A case report and review of the literature

BACKGROUNDAnaplastic large cell lymphoma (ALCL) is a rare and heterogeneous malignant tumor, which is classified as anaplastic lymphoma kinase (ALK)positive ALCL and ALK- ALCL. Many patients are diagnosed with ALCL at the stage of bone marrow involvement. However, ALCL patients with clinical manifestations consistent with acute leukemia are relatively rare. CASE SUMMARYIn this report, the patient did not receive appropriate diagnosis and treatment despite a two-year history of lymph node enlargement. Hereafter, she was admitted for B symptoms and was diagnosed as ALK-ALCL by lymph node biopsy. Then, the disease progressed to leukemia without any treatment after 2 mo. The proportion of lymphoma cells in bone marrow was as high as 96%, and the proportion of peripheral blood was 84%. She also had clinical manifestations similar to acute leukemia. After completion of chemotherapy, she developed granulocytopenia and fever and died from septicemia.CONCLUSIONALCL with leukemic presentation is a late manifestation of lymphoma with low chemotherapy tolerance and poor prognosis.

The novel ALK inhibitor ZX-29 induces apoptosis through inhibiting ALK and inducing ROS-mediated endoplasmic reticulum stress in Karpas299 cells.

It is a well-known fact that 60%-85% of anaplastic large cell lymphoma (ALCL) is mainly driven by the anaplastic lymphoma kinase (ALK) fusion protein. Although ALK-positive ALCL patients respond significantly to ALK inhibitors, the development of resistance is inevitable, which requires the development of new therapeutic strategies for ALK-positive ALCL. Here, we investigated the anticancer activities of N-(2((5-chloro-2-((2-methoxy-6-(4-methylpiperazin-1-yl)pyridin-3yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (ZX-29), a newly synthesized ALK inhibitor, againstnucleophosmin-ALK-positive cell line Karpas299. We demonstrated that ZX-29 decreased Karpas299 cells growth and had better cytotoxicity than ceritinib, which was mediated through downregulating the expression of ALK and related proteins, inducing cell cycle arrest, and promoting cell apoptosis. Moreover, ZX-29-induced cell apoptosis by inducing endoplasmic reticulum stress (ERS). In addition, ZX-29 increased the generation of reactive oxygen species (ROS), and cells pretreatment with N-acetyl- l-cysteine could attenuate ZX-29-induced cell apoptosis and ERS. Taken together, ZX-29 inhibited Karpas299 cell proliferation and induced apoptosis through inhibiting ALK and its downstream protein expression and inducing ROS-mediated ERS. Therefore, our results provide evidence for a novel antitumor candidate for the further investigation.

Abstract PO-50: Phophatases modulate resistance to ALK inhibitors in anaplastic large-cell lymphoma

Abstract Introduction: Anaplastic large-cell lymphomas (ALCL) frequently carry oncogenic fusions involving the anaplastic lymphoma kinase (ALK) gene. The ALK fusions activate several signaling pathways, promoting cell growth, migration, and survival. Chemotherapy is the standard treatment for ALCL patients, but about 30% of patients relapse. Targeting ALK using tyrosine kinase inhibitors (TKIs) has shown promising results, and the FDA recently granted breakthrough therapy designation to crizotinib for use in patients with relapsed/refractory ALK+ ALCL. However, resistance to crizotinib develops in ALK+ ALCL patients secondary to ALK mutations or unknown mechanisms. In this study, we aimed at elucidating unknown bypass mechanisms of crizotinib resistance in ALK+ ALCL. Methods: We used Genome-wide CRISPR-Cas9 Knockout Screening (GeCKO.v2) to identify candidate genes that contribute to resistance to crizotinib. Four different ALCL cell lines were infected with Lenti-GeCKO libraries. After treatment with crizotinib for 14 days to select for resistant cells, next-generation sequencing was performed on crizotinib-resistant cells to identify candidate resistance genes. Top candidates were selected for validation assays and further analyses. Results: Genomic loss-of-function screens identified two phosphatases, PTPN1 and PTPN2, in all ALCL cell lines as consistent top hits driving resistance to crizotinib. Functional validation of these candidate genes showed that single loss of either PTPN1 or PTPN2 generates immediate resistance to crizotinib and other ALK TKIs such as alectinib and lorlatinib. Consistently, RNA-seq in patients who developed resistance to crizotinib showed downregulation of PTPN1 or PTPN2 expression. By multiple assays, we demonstrate that PTPN1 and PTPN2 are phosphatases that de-phosphorylate ALK, thereby regulating its overall phosphorylation levels and activity. In addition, we found that PTPN1, but not PTPN2, is also a phosphatase of SHP2, a key mediator of oncogenic ALK signaling. Downstream signaling analysis showed that deletion of PTPN1 or PTPN2 induces resistance to crizotinib by hyperactivating the MAPK and JAK/STAT pathways. A treatment that combined crizotinib and a recently developed SHP2 inhibitor (SHP099) completely blocked the sustained MAPK activation and reverted crizotinib resistance in vitro and in vivo. Conclusions: We discovered that PTPN1 and PTPN2 are ALK phosphatases that control sensitivity to ALK TKIs in ALCL. Combined inhibition of SHP2 is a potential therapeutic approach to overcome resistance to ALK TKIs in ALCL. Citation Format: Elif Atabay, Qi Wang, Taek-Chin Cheong, Nina Prokoph, Chiara Ambrogio, Ines Mota, Achille Pich, Enrico Patrucco, Claudia Voena, Roberto Chiarle. Phophatases modulate resistance to ALK inhibitors in anaplastic large-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-50.

A Case of Central Nervous System Metastasis of Anaplastic Lymphoma Kinase Positive Anaplastic Large Cell Lymphoma Manifested as Central Nervous System Vasculitis

Introduction: Anaplastic Large Cell Lymphoma (ALCL) is a rare distinct subset of T-cell non-Hodgkin lymphomas (T-cell NHL). Anaplastic Lymphoma Kinase (ALK) positive ALCL patients are generally associated with younger age and have a better prognosis. While Central Nervous System (CNS) involvement in T-cell NHL is rare and has a dismal prognosis. There was no report about ALK + ALCL and CNS vasculitis. Case presentation: A 17-year-old boy who was diagnosed ALK + ALCL and underwent six courses of systemic chemotherapy presented as severe CNS vasculitis in the beginning, but eventually he was diagnosed CNS metastasis of ALCL by the presence of cranial nerve palsy, the demonstration of lesions on MRI and PET-CT, and Flow cytometry detection of tumor cells in Cerebrospinal Fluid (CSF). Conclusion: We first present a case of CNS metastasis of systemic ALK + ALCL accompanied with CNS vasculitis. When an ALK + ALCL patient had CNS symptoms while imaging revealed CNS vasculitis, we need to be vigilant that the patient may have CNS involvement.

TCL-302: Immunohistochemical Characterization of Anaplastic Large Cell Lymphoma Using Tissue Microarray

Context: Anaplastic Large Cell Lymphoma (ALCL) is a type of mature T-cell lymphomas with systemic and primary cutaneous (PCALCL) subtypes. Systemic ALCL includes anaplastic lymphoma kinase (ALK)+ and ALK-. ALK+ usually has the NPM/ALK gene, which has favorable prognosis compared to ALK-. PCALCL is usually indolent. DUSP22 rearrangement in ALK-ALCL has a favorable outcome like ALK+, indicating alternate pathways of ALCL. Objective: Our primary aim was to compare protein expression levels of selected signaling molecules in oncogenic pathways of ALCL (JAK1, STAT3, DUSP22, ERB4, BLIMP1, SOCS3) among the three subtypes. We also correlated expression levels to ORR, PFS, DFS, and OS. Design: This is a retrospective study correlating protein expression levels of various antibodies across 27 patient tissue samples. Setting: The study was set at H. Lee Moffitt Cancer Center and Research Institute, an academic referral center. Patients or other participants: We identified 50 ALCL patients from 2000–2019. Twenty-seven tissue samples (6 ALK+, 10 ALK-, 8 PCALCL, 3 controls) met eligibility criteria, which included pathologist-reviewed diagnosis of ALCL subtypes (ALK+, ALK-, PCALCL) and consent to the Tissue Core database. Interventions: Replicate cores were taken and TMAs were constructed from formalin-fixed, paraffin-embedded lymph node or skin biopsies. IHC was performed with Abs to selected proteins. Stained TMA was scanned using Aperio™ ScanScope XT2 with a 200×/0.8NA objective lens at 5 min/slide via Basler tri-linear array detection. TMAs were analyzed using Aperio Nuclear V9 Default algorithm with thresholds for stain intensity: Strong = 130, Moderate = 188, Weak = 162 to determine the % positive biomarker stain within each core. Main outcome measures: The primary study outcome measurement was comparison of protein expression levels based on specific pathways known or hypothesized to be involved in ALCL pathogenesis. We hypothesized that these protein expression levels would correlate to time to event outcomes Results: PCALCL, ALK+, and ALK- subgroups demonstrated a significant difference in SOCS3 expression with mean and std respectively: 42.4% (0.153), 17.4% (0.089), and 21.0% (0.155);p=0.008. Among all pairs, ALK+ and PCALCL were statistically different (p=0.011). Patients with high BLIMP1 expression (% larger than median, 28.5%) regardless of ALCL subtype were associated with better mOS (not reached) compared to patients with low ( Conclusions: We identified a significant difference in expression of two Abs among three subtypes of ALCL. Increased BLIMP1 was associated with favorable prognosis and could potentially be a positive prognostic marker. Increased SOCS3 appears more prevalent in PCALCL than systemic ALCL, suggesting a potential utility in DDx. We plan to validate our results in a larger sample. Adding secondary markers like Bcl-2 can be used in the future to correlate expression levels and response to targeted agents, such as venetoclax. Funding: University of South Florida Graduate Medical Education Grant ($7500).

Abstract 4715: Characterization of potential co-drivers of pathogenesis in ALK positive ALCL

Abstract ALK positive Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma characterized by expression of the NPM/ALK fusion tyrosine kinase essential for neoplastic cells growth. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. Although ALK+ ALCL cells are mainly driven by the constitutively active NPM/ALK kinase, presence of additional mutations might affect the disease course and response to treatment, leading to different outcomes in different patients. We employed whole exome sequencing to characterize the co-mutational burden in ALK+ ALCL samples at diagnosis. Diagnosis biopsy and matched healthy control DNA samples from 24 ALK+ ALCL patients were sequenced. Somatic variants present at high frequency within the tumor (at least 25% variant allele frequency), which can be viewed as potential co-drivers, were annotated; the identified mutations were weighed using in silico tools to predict their oncogenic potential and their effects on protein function. All identified variants were confirmed by Sanger sequencing. Gene ontology and pathway analyses identified cell adhesion and actin cytoskeleton among the most enriched biological functions. Using the above-mentioned criteria, we found recurrent somatic variants in 7 genes including PPP1R9A, RUNX1T1, FAT4 and RBBP8. Here we report the initial characterization of the role of FAT4 in ALCL cells. FAT4 is a member of the cadherin superfamily with tumor suppressor functions. In our cohort, 3/24 patients (13%) were found to carry FAT4 mutations, including nonsense mutations and missense changes predicted to be deleterious, suggesting a loss of function effect. To characterize the biological consequences of these mutations, we generated FAT4 knockdown ALCL cell lines. FAT4-silenced cells grew faster and showed increased migratory properties compared to parental cells. They also differed from parental cells in terms of morphology, having bigger size and modulated F-actin rearrangement. Tumor suppressing activity of FAT4 has been linked to Wnt/β-catenin pathway; we found that FAT4-silenced cells had decreased levels of serine/threonine phosphorylated β-catenin and higher nuclear localization, which insinuates activation of Wnt/β-catenin signalling and henceforth, increased cellular proliferation. To explore therapeutic implications of FAT4 mutations, we analyzed the sensitivity of FAT4 silenced cells to chemotherapeutic agents. FAT4 knockdown cells were less sensitive to a CHOP combination treatment as compared to parental cells. No difference was seen in terms of sensitivity to crizotinib. Finally, we investigated the effects of RUNX1T1 somatic variants found in our study. Preliminary luciferase assay results showed that the variants significantly increase the transcriptional repression ability of RUNX1T1. Altogether, our data provides information about genes other than NPM-ALK that might play a role in the pathogenesis of ALCL. Citation Format: Luca Mologni, Geeta G. Sharma, Matteo Villa, Mario Mauri, Giulia Arosio, Cosimo Lobello, Hugo Larose, Alessandra Pirola, Silvia Bombelli, Nicoletta Cordani, Luca Massimino, Šárka Pospíšilová, Suzanne D. Turner, Giorgio Inghirami, Fabio Pagni, Rocco Piazza, Roberto Perego, Roberto Chiarle, Carlo Gambacorti-Passerini. Characterization of potential co-drivers of pathogenesis in ALK positive ALCL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4715.

Decreased MYC-associated factor X (MAX) expression is a new potential biomarker for adverse prognosis in anaplastic large cell lymphoma

MYC-associated factor X (MAX) is a protein in the basic helix-loop-helix leucine zipper family, which is ubiquitously and constitutively expressed in various normal tissues and tumors. MAX protein mediates various cellular functions such as proliferation, differentiation, and apoptosis through the MYC-MAX protein complex. Recently, it has been reported that MYC regulates the proliferation of anaplastic large cell lymphoma. However, the expression and function of MAX in anaplastic large cell lymphoma remain to be elucidated. We herein investigated MAX expression in anaplastic large cell lymphoma (ALCL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) and found 11 of 37 patients (30%) with ALCL lacked MAX expression, whereas 15 of 15 patients (100%) with PTCL-NOS expressed MAX protein. ALCL patients lacking MAX expression had a significantly inferior prognosis compared with patients having MAX expression. Moreover, patients without MAX expression significantly had histological non-common variants, which were mainly detected in aggressive ALCL cases. Immunohistochemical analysis showed that MAX expression was related to the expression of MYC and cytotoxic molecules. These findings demonstrate that lack of MAX expression is a potential poor prognostic biomarker in ALCL and a candidate marker for differential diagnosis of ALCL and PTCL-NOS.

Whole Exome Sequencing reveals NOTCH1 mutations in anaplastic large cell lymphoma and points to Notch both as a key pathway and a potential therapeutic target.

Patients diagnosed with anaplastic large cell lymphoma (ALCL) are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. To understand disease pathology and to uncover novel targets for therapy, we performed whole-exome sequencing of anaplastic lymphoma kinase (ALK)+ ALCL, as well as gene-set enrichment analysis. This revealed that the T-cell receptor and Notch pathways were the most enriched in mutations. In particular, variant T349P of NOTCH1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK– ALCL patients’ samples. Furthermore, we demonstrated that NPM-ALK promotes NOTCH1 expression through binding of STAT3 upstream of NOTCH1. Moreover, inhibition of NOTCH1 with γ- secretase inhibitors or silencing by short hairpin RNA leads to apoptosis; cotreatment in vitro with the ALK inhibitor crizotinib led to additive/synergistic antitumor activity suggesting that this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, crizotinib-resistant and -sensitive ALCL were equally sensitive to γ-secretase inhibitors. In conclusion, we show a variant in the extracellular domain of NOTCH1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.

RNY4 in Circulating Exosomes of Patients With Pediatric Anaplastic Large Cell Lymphoma: An Active Player?

Emerging evidence indicates that extracellular vesicles, particularly exosomes, play a role in several biological processes and actively contribute to cancer development and progression, by carrying and delivering proteins, transcripts and small RNAs (sRNAs). There is high interest in studying exosomes of cancer patients both to develop non-invasive liquid biopsy tests for risk stratification and to elucidate their possible involvement in disease mechanisms. We profiled by RNA-seq the sRNA content of circulating exosomes of 20 pediatric patients with Anaplastic Large Cell Lymphoma (ALCL) and five healthy controls. Our analysis disclosed that non-miRNA derived sRNAs constitute the prominent fraction of sRNA loaded in exosomes and identified 180 sRNAs significantly more abundant in exosomes of ALCL patients compared to controls. YRNA fragments, accounting for most of exosomal content and being significantly increased in ALCL patients, were prioritized for further investigation by qRT-PCR. Quantification of RNY4 fragments and full-length sequences disclosed that the latter are massively loaded into exosomes of ALCL patients with more advanced and aggressive disease. These results are discussed in light of recent findings on the role of RNY4 in the modulation of tumor microenvironment.

Outcomes of 33 patients with anaplastic large cell lymphoma treated after hematopoietic stem cell transplantation

目的探讨造血干细胞移植治疗间变大细胞淋巴瘤（ALCL）的疗效及预后。方法回顾性分析全国八家三甲医院2005年1月至2017年12月收治的33例接受造血干细胞移植（HSCT）的ALCL患者临床资料，评价自体造血干细胞移植（auto-HSCT）和异基因造血干细胞移植（allo-HSCT）治疗ALCL的生存率、复发率和影响预后的相关因素。结果33例接受HSCT的ALCL患者的中位发病年龄为31（12~57）岁，男23例，女10例，间变性淋巴瘤激酶阳性（ALK+）和阴性（ALK−）分别为24例（72.7%）和9例（27.3%）。25例患者接受auto-HSCT（ALK+患者19例，ALK−患者6例），8例患者接受allo-HSCT（ALK+患者5例，ALK−患者3例）。移植后中位随访时间18.7（4.0~150.0）个月。移植前疾病缓解状态：完全缓解6例（均行auto-HSCT），部分缓解16例（auto-HSCT组14例，allo-HSCT组2例），复发难治11例（auto-HSCT组5例，allo-HSCT组6例）。疾病进展死亡7例，其中auto-HSCT组5例（20.0%），allo-HSCT组2例（25.0%）。移植相关死亡（TRM）5例，其中auto-HSCT组2例（8.0%），allo-HSCT组3例（37.5%）。auto-HSCT后中位无进展生存（PFS）和总生存（OS）时间均为15个月，allo-HSCT后中位PFS时间为3.7（1.0~90.0）个月，中位OS时间为4.6（1.0~90.0）个月，两组生存曲线差异无统计学意义（OS及PFS P值分别为0.247和0.317）。auto-HSCT和allo-HSCT组的2年OS率分别为72%和50%，5年OS率分别为36%和25%。结论ALCL化疗反应率高，有不良预后因素的情况下化疗后序贯auto-HSCT为重要治疗措施，高危患者或可从allo-HSCT中获益。

KRCA-0008 suppresses ALK-positive anaplastic large-cell lymphoma growth.

Anaplastic lymphoma kinase (ALK), which belongs to the insulin receptor tyrosine kinase superfamily, plays an important role in nervous system development. Due to chromosomal translocations, point mutations, and gene amplification, constitutively activated ALK has been implicated in a variety of human cancers, including anaplastic large-cell lymphoma (ALCL), non-small cell lung cancer, and neuroblastoma. We evaluated the anti-cancer activity of the ALK inhibitor KRCA-0008 using ALCL cell lines that express NPM (nucleophosmin)-ALK. KRCA-0008 strongly suppressed the proliferation and survival of NPM-ALK-positive ALCL cells. Additionally, it induced G0/G1 cell cycle arrest and apoptosis by blocking downstream signals including STAT3, Akt, and ERK1/2. Tumor growth was strongly suppressed in mice inoculated with Karpas-299 tumor xenografts and orally treated with KRCA-0008 (50mg/kg, BID) for 2weeks. Our results suggest that KRCA-0008 will be useful in further investigations of ALK signaling, and may provide therapeutic opportunities for NPM-ALK-positive ALCL patients.

18F-FDG PET/CT imaging findings in anaplastic large cell lymphoma, a rare subtype of lymphoma

ObjectiveTo investigate the 18F-FDG PET/CT imaging manifestations for anaplastic large cell lymphoma (ALCL), a rare subtype of T/NK cell lymphoma.MethodsFifty patients with ALCL, including 32 anaplastic lymphoma kinase (ALK)-positive patients and 18 ALK-negative patients, were enrolled. The positive detection, maximal standardized uptake value (SUVmax), and distribution of nodal and extranodal involvement were recorded and analysed. Fifty patients with diffuse large B cell lymphoma (DLBCL) were collected as a control group.ResultsALCL lesions were demonstrated to be 18F-FDG-avid tumours with a mean SUVmax of 19.4 ± 12.6. Most (76%) ALCL patients presented with stage III-IV disease, and nodal and extranodal involvement occurred in 74.0 and 72.0% of the patients, respectively. ALCL and DLBCL showed many similarities in tumour stage, 18F-FDG uptake and tumour involvement (P > 0.05), although the preferred extranodal organs of involvement (bone and the gastrointestinal tract, respectively) were different (P < 0.05). Compared to ALK-negative lesions, a higher uptake of 18F-FDG was found in the ALK-positive lesions (SUVmax: 22.1 ± 14.3 vs. 15.1 ± 6.6, t = 2.354, P = 0.023). ALK-positive ALCL was more likely to involve the lymph nodes than ALK-negative ALCL (84.3% vs. 55.5%, χ2 = 4.973, P = 0.043), while ALK-negative ALCL was more prone to involve the extranodal organs compared to ALK-positive ALCL (88.9% vs. 62.5%, χ2 = 3.979, P = 0.046).ConclusionThe present study demonstrated that ALCL is a systemic 18F-FDG-avid lymphoma with many imaging manifestations similar to DLBCL on PET/CT. The present study also showed that ALK expression actually influenced tumour 18F-FDG uptake and lesion distribution. These findings may be useful to improve the understanding of the biological characteristics of ALCL.

Favorable outcomes and reduced toxicity with a novel vinblastine-based non-high dose methotrexate (HDMTX) regimen (modified MCP-842) in pediatric anaplastic large cell lymphoma (ALCL): experience from India

Anaplastic large cell lymphoma (ALCL) is a rare form of non-Hodgkin lymphoma (NHL) in children. Most treatment regimens include high-dose methotrexate (HDMTX), which is logistically difficult to administer in resource-limited settings. We evaluated the outcomes of pediatric ALCL patients treated on a uniform protocol (Modified Multicentric Protocol, MCP-842 regimen) at our hospital between January 2005 and December 2016. Of the 68 patients who received treatment on the Modified MCP842 protocol, 46 patients are alive in remission, 11(16%) had disease progression, 9(13%) relapsed after achieving remission, and 5(7%) had treatment-related mortality (TRM). Seventeen of 20 relapsed/progressed patients subsequently expired. With a median follow-up of 55 months (range 2–165 months), the 4-year event-free survival (EFS) and overall survival (OS) are 63% (95% CI of 50–73%) and 70%(95% CI of 57–79%), respectively. An indigenous protocol using vinblastine (without HDMTX and steroids) is feasible in a resource-limited setting and achieves outcomes comparable to regimens incorporating HDMTX, with lower toxicity.