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

Anaplastic large cell lymphomas with the 6p25.3 rearrangement are a heterogeneous group of tumours with a diverse molecular background

ALK-negative anaplastic large cell lymphoma (ALCL) cases with 6p25.3 rearrangement are characterized by peculiar morphological and immunohistochemical features compare to 6p25.3-negative ALK-negative ALCL cases. A subgroup of 6p25.3-positive ALK-negative ALCL cases show the t(6,7) (p25.3;q32.3) rearrangement. Aims: To analyse the differences between 6p25.3-rearranged cases with and without t(6,7) (p25.3;q32.3). Using RNA-sequencing we studied a series of 17 samples showing 6p25.3-rearrangement, identified by FISH, consisting of seven systemic and eight primary cutaneous cases including two examples of secondary skin involvement by systemic ALCL. RNA-sequencing exclusively detected a translocation involving a gene in the 6p25.3 region (either IRF4 or DUSP22) in 7/14 cases (50%). In six of these seven cases the partner proved to be the LINC-PINT region in chromosome 7, while an EXOC2::DUSP22 rearrangement was found in one case. All cases but one were primary cutaneous ALCLs. They all were CD3 positive and BCL2 negative, while most of them expressed p-STAT3. On the contrary, cases without the t(6,7) (p25.3;q32.3) were mainly systemic (71%, 5/7) against just two pcALCL. In general, they lose CD3 (50% positive) and p-STAT3 (25% positive) expression, being all of them BCL2 positive. Moreover, in 60% of them other gene fusions were found. At the transcriptional level, they were characterized by the overexpression of TCF3 (TCF7L1/E2A), DLL3, CD58 and BCL2 genes 75%(6/8) of pcALCL with 6p25.3 rearrangement featured the so-called "biphasic morphologic pattern, which was not found in cutaneous involvement from systemic ALCL. 83% (5/6) of the pcALCL cases with the "biphasic morphologic pattern" showed the t(6,7) (p25.3;q32.3) rearrangement. ALK-negative ALCL cases with 6p25.3 rearrangement are a subgroup of tumours that are heterogeneous with respect to the presence or absence of the t(6,7) (p25.3;q32.3) translocation.

DUSP22 rearrangement is associated with a distinctive immunophenotype but not outcome in patients with systemic ALK-negative anaplastic large cell lymphoma.

DUSP22 rearrangement (R) has been associated with a favorable outcome in systemic ALK-negative anaplastic large cell lymphoma (ALCL). However, a recent study found that patients with DUSP22-R ALK-negative ALCL have a poorer prognosis than was reported initially. In this study, we compared the clinicopathological features and outcomes of patients with ALKnegative ALCL with DUSP22-R (n=22) versus those without DUSP22-R (DUSP22-NR; n=59). Patients with DUSP22-R ALCL were younger than those with DUSP22-NR neoplasms (P=0.049). DUSP22-R ALK-negative ALCL cases were more often positive for CD15, CD8, and less frequently expressed pSTAT3Tyr705, PD-L1, granzyme B and EMA (all P<0.05). TP63 rearrangement (TP63-R) was detected in three of the 66 (5%) ALK-negative ALCL cases tested and none of these cases carried the DUSP22-R. Overall survival of patients with DUSP22-R ALCL was similar to that of the patients with DUSP22-NR neoplasms regardless of International Prognostic Index score, stage, age, or stem cell transplantation status (all P>0.05), but was significantly shorter than that of the patients with ALK-positive ALCL (median overall survival 53 months vs. undefined, P=0.005). Five-year overall survival rates were 40% for patients with DUSP22-R ALCL versus 82% for patients with ALK-positive ALCL. We conclude that DUSP22-R neoplasms represent a distinctive subset of ALK-negative ALCL. However, in this cohort DUSP22-R was not associated with a better clinical outcome. Therefore, we suggest that current treatment guidelines for this subset of ALK-negative ALCL patients should not be modified at present.

ALK-negative anaplastic large cell lymphoma with DUSP22 rearrangement has distinctive disease characteristics with better progression-free survival: a LYSA study.

ALK-negative anaplastic large cell lymphoma (ALCL) comprises subgroups harboring rearrangements of DUSP22 (DUSP22- R) or TP63 (TP63-R). Two studies reported 90% and 40% 5-year overall survival (OS) rates in 21 and 12 DUSP22-R/TP63- not rearranged (NR) patients, respectively, making the prognostic impact of DUSP22-R unclear. Here, 104 newly diagnosed ALK-negative ALCL patients (including 37 from first-line clinical trials) from the LYSA TENOMIC database were analyzed by break-apart fluorescence in situ hybridization assays for DUSP22-R and TP63-R. There were 47/104 (45%) DUSP22-R and 2/93 (2%) TP63-R cases, including one DUSP22-R/TP63-R case. DUSP22-R tumors more frequently showed CD3 expression (62% vs. 35%, P=0.01), and less commonly a cytotoxic phenotype (27% vs. 82%; P<0.001). At diagnosis, DUSP22- R ALCL patients more frequently had bone involvement (32% vs. 13%, P=0.03). The patient with DUSP22-R/TP63-R ALCL had a rapidly fatal outcome. After a median follow-up of 4.9 years, 5-year progression-free survival (PFS) and OS rates of 84 patients without TP63-R treated with curative-intent anthracycline-based chemotherapy were 41% and 53%, respectively. According to DUSP22 status, 5-year PFS was 57% for 39 DUSP22-R versus 26% for 45 triple-negative (DUSP22-NR/TP63-NR/ALK-negative) patients (P=0.001). The corresponding 5-year OS rates were 65% and 41%, respectively (P=0.07). In multivariate analysis, performance status and DUSP22 status significantly affected PFS, and distinguished four risk groups, with 4-year PFS and OS ranging from 17% to 73% and 21% to 77%, respectively. Performance status but not DUSP22 status influenced OS. The use of brentuximab vedotin in relapsed/refractory patients improved OS independently of DUSP22 status. Our findings support the biological and clinical distinctiveness of DUSP22- R ALK-negative ALCL. Its relevance to outcome in patients receiving frontline brentuximab vedotin remains to be determined.

ALK-Negative Anaplastic Large Cell Lymphoma (ALCL): Prognostic Implications of Molecular Subtyping and JAK-STAT Pathway.

The anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a clinically distinct but heterogeneous entity and lacks the specific immunophenotypic or genetic features compared with the ALK-positive ALCL. Recent molecular studies have provided genetic landscapes of ALK-negative ALCL that have prognostic significance. In this study, we subtyped ALK-negative ALCL based on DUSP22 rearrangements and TP63 expression and also looked for mutations in JAK-STAT pathway. The subtyping of the ALK-negative ALCL in relation to DUSP22 rearrangement and TP63 expression was done using fluorescence in situ hybridization and immunohistochemistry, respectively. The hotspot JAK-STAT mutations were analyzed using Sanger sequencing and amplification refractory mutation system polymerase chain reaction (PCR) and Signal transducer and activator of transcription 3 (STAT3) expression by immunohistochemistry. Forty-eight cases of ALCL were included with median age of 30 years and sex ratio of 1.8:1. The p63 expression was detected in 26.7% of ALK-negative ALCL cases. DUSP22 rearrangement was noted in 12.5% cases of p63-negative ALK-negative ALCLs. DUSP22 rearranged cases had better overall survival in contrast to p63 expressing and triple negative ALCLs. Triple negative ALCLs showed inferior overall survival rate. STAT3 expression was evident in 61.1% and 60% of ALK-positive and ALK-negative ALCLs, respectively. None of the cases subjected to Sanger sequencing as well as amplification refractory mutation system PCR for hotspot mutation analysis of JAK1 (exon 24) and STAT3 (exon 21) revealed any mutation. ALK-negative ALCL is a genetically heterogeneous disease with widely disparate clinical outcomes. Subtyping of ALK-negative ALCL based on DUSP22 rearrangement and p63 expression provides prognostic information.

Leukemic phase of ALK-negative anaplastic large cell lymphoma in a patient who is on androgenic steroids: A case report.

IntroductionALK-negative anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma that usually involves lymph nodes or extranodal sites. Leukemic phase of ALK-negative ALCL is exceedingly rare and often carries a poor prognosis. Androgenic steroids have gained popularity among the young, and at higher doses, it can result in immune dysregulation and may be potentially carcinogenic. Case presentation: A 30-year-old gentleman of Malay ethnicity presented to the hematology department with night fevers, loss of weight and bony pain for the past 6 weeks. He is a gymnasium instructor with a history of chronic usage of intramuscular testosterone enanthate. Physical examination revealed ecchymosis over the left elbow and hepatomegaly. A complete blood count depicted anemia, thrombocytopenia and leucocytosis. An 18-Fluorodeoxyglucose positron emission tomography (18-FDG PET/CT) imaging showed a hypermetabolic anterior mediastinal mass of 6.8 × 7.0 × 6.5 cm with diffuse hypermetabolism in the liver, spleen and axial skeleton. The bone marrow trephine and mediastinal tissue histology were consistent with leukemic ALK-negative ALCL. He was treated with CHOEP (cyclophosphamide, doxorubicin, vincristine, etoposide, prednisolone) induction chemotherapy in which he required intensive antibiotic and blood support. He progressed with worsening B symptoms and new diffuse lymphadenopathies suggesting rapid dissemination of the disease. He subsequently succumbed to multiorgan failure with disseminated intravascular coagulopathy at the intensive care unit. Conclusion: Leukemic phase ALK-negative ALCL often carries a complex karyotype and requires early intensive polychemotherapy. Use of anabolic steroids depletes the ability of defending lymphocytes to remove tumour producing cells.

FOXP3-positive T-cell lymphomas in non-HTLV1 carriers include ALK-negative anaplastic large cell lymphoma: expanding the spectrum of T-cell lymphomas with regulatory phenotype

Forkhead box P3 (FOXP3) is a specific marker for regulatory T-cells (Tregs). We report 6 cases of T-cell lymphomas with Treg phenotype based on diffuse positivity for FOXP3 in tumor cells. The patients showed a median age of 56 years with a male predominance. Sites of disease included lymph nodes (4), skin (2), subcutaneous tissue (1) and bone marrow (1). All cases showed monomorphic large cells, some with Hodgkin-like or anaplastic cells. All cases expressed pan T-cell markers and lacked cytotoxic markers; one case showed diffuse PD1 staining. Only one case harbored human T-lymphotrophic virus (HTLV)-1 DNA within tumor cells and was classified as adult T-cell leukemia/lymphoma (ATLL). Among 5 HTLV1-negative cases, 3 were classified as peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) and 2 fulfilled criteria for ALK-negative anaplastic large cell lymphoma (ALCL) with diffuse and strong CD30 positivity. We concluded that Treg phenotype may be rarely seen in HTLV1-negative cases, such as PTCL, NOS and ALK-negative ALCL. Our findings expand the spectrum of T-cell lymphomas with regulatory phenotype and suggest that consideration should be given to HTLV1 DNA testing in the appropriate clinical setting to rule out ATLL.

Elevated serum interleukin-10 level and M2 macrophage infiltration are associated with poor survival in angioimmunoblastic T-cell lymphoma.

Interleukin-10 (IL-10) induces an immunosuppressive microenvironment including M2 macrophages, inhibiting anti-tumor immunity. The aim of this study was to evaluate whether serum IL-10 level at diagnosis and tissue infiltration of M2 macrophages could predict survival outcome of patients with angioimmunoblastic T-cell lymphoma (AITL).We measured serum levels of IL-5, IL-10, IL-12, and interferon-gamma (IFN-γ) at diagnosis in AITL and other common subtypes of nodal T-cell lymphoma including peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), ALK-negative anaplastic large cell lymphoma (ALCL) or ALK-positive ALCL between September 2008 and December 2014. We also analyzed the infiltration of CD68- and CD163-positive macrophages in tumor tissue of AITL. In total, 97 patients with AITL (n=37), PTCL-NOS (n=40), ALK-negative ALCL (n=11), or ALK-positive ALCL (n=9) were treated with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Among cytokines, only the serum level of IL-10 was significantly higher in AITL patients than in other subtypes (P < 0.05). Compared to other subtypes, the association of serum IL-10 with overall survival (OS) was only significant in AITL. Accordingly, the response to CHOP chemotherapy was significantly worse in the high IL-10 group, and infiltration of CD163-positive M2 macrophages was significantly associated with OS in AITL. In conclusion, this study demonstrated the prognostic relevance of serum IL-10 and tissue infiltration of M2 macrophages in AITL patients. Our results suggest the possible use of these variables as potential therapeutic targets and novel prognostic indicators in patients with AITL.

T-Cell Lymphoma Patient-Derived Xenografts and Newly Developed Cell Lines Recapitulate Aspects of Disease Biology and Represent Novel Tools for Preclinical Drug Development

Lymphomas represent nearly 70 distinct diseases with unique clinical presentations, therapeutic responses and underlying biology. There is a pressing shortage of publically available cell line and in vivo models of nearly all of these diseases; T-cell lymphoma models are particularly under-represented compared to B-cell lymphomas, which has severely hampered efforts to understand and target their biology. The majority ofin vivo models of T-cell lymphomas are genetically-engineered mouse models, which often don't faithfully recapitulate human disease. To address this issue, we have established a repository of patient-derived xenografts (PDX) of lymphomas by engrafting human tumors into immunodeficient NOD/Scid/IL2rgnull mice with or without an MHC Class 1 deficiency (to prevent graft versus host disease). Blood and bone marrow specimens involved with tumor were injected by tail vein injection. Lymph node and extranodal biopsy specimens were implanted under the renal capsule as a 1x1x2mm tumor seed, which maintains the in situ microarchitecture. A description of T-cell lymphoma PDXs is included in the Table.PDXs have been extensively characterized by immunohistochemistry (IHC), flow cytometry, transcriptome sequencing and targeted DNA sequencing. These studies have demonstrated retention of key architectural, cellular, and molecular features of the primary tumors. Flow cytometric analysis of patient tumors and their respective xenografts revealed highly concordant patterns of surface marker expression. IHC of murine tissues confirmed retention of tumor immunophenotypes, architecture, and even tissue tropism in the PDXs. For example, blood from a patient with Sézary Syndrome manifested in the skin of recipient mice when injected into the lateral tail vein. A breast implant-associated ALK-negative anaplastic large cell lymphoma (ALCL) implanted under the renal capsule metastasized to the liver and spleen while uniformly retaining CD30 positivity. A peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) specimen implanted under the renal capsule engrafted in the spleen, with the notable admixture of nonmalignant T cells and scattered EBV-positive B cells in first passage. T-cell receptor gene rearrangement PCR performed on this PTCL-NOS demonstrated an identical rearrangement pattern in the primary tumor and the PDX. An angioimmunoblastic T-cell lymphoma (AITL) specimen engrafted in spleen, lymph node and bone marrow within 6 weeks and serially transplanted through three generations in an orthotopic manner while maintaining a CD3+CD4+PD1+CD30partial immunophenotype.The genetic characterization of the PDX models using a targeted DNA sequencing approach showed a mutational profile that clearly matched primary T-cell lymphoma samples and significantly expands the current repertoire of available pre-clinical models. For example, a PDX model of AITL showed mutations of TET2 and ARID1B; a model of an ALK-negative ALCL harbored mutations of STAT3 and STAT5. This massively extends the spectrum of clinically representative model systems that can be used to explore novel therapeutic strategies for T-cell lymphomas. Several early-passage PDXs have been used to generate T-cell lymphoma cells lines, including three cell lines from AITL PDX models. One of these AITL cell lines has proliferated through 30 passages and was validated by immunophenotype and molecular confirmation of bi-allelic TET2 mutations with loss of 6q, 7q, and 10q confirmed using Sanger and TruSeq Custom Amplicon Sequencings. To our knowledge, there have been no reports of an AITL cell line in the literature. Additional peripheral T-cell lymphoma cell lines are currently under development.These lymphomas, along with a spectrum of PDXs of other hematologic malignancies, are available to collaborators through the online portal PRoXe (Public Repository of Xenografts) at www.proxe.org. These models represent a unique opportunity to interrogate biology and perform preclinical studies with in vivo models. [Display omitted] DisclosuresJacobson:Kite: Membership on an entity's Board of Directors or advisory committees. Armand:Pfizer: Research Funding; Sequenta Inc: Research Funding; Merck: Consultancy, Research Funding; Roche: Research Funding; Infinity Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding. Shipp:Bristol-Myers Squibb: Consultancy, Research Funding; Cell Signaling: Honoraria; Merck, Gilead, Takeda: Other: Scientific Advisory Board; Bayer: Research Funding. Fisher:Pharmacyclics: Consultancy. Weinstock:Novartis: Consultancy, Research Funding.

Axillary Lymphadenopathy

Till date, there is only one reported case of breast implant-associated ALK-negative anaplastic large cell lymphoma (ALCL) with an axillary presentation that followed an aggressive behavior. We report the case of a 50-year-old female presenting with an axillary lymphadenopathy 8 years after breast prostheses implantation. Clinical examination, ultrasound, and magnetic resonance imaging detected no mammary lesions. The lymph node showed intrasinusoidal infiltration by large pleomorphic cells expressing CD30 and lacking ALK-immunoreactivity. Tumor staging was negative. Cells with identical features were found in the ipsilateral periprosthetic capsule. The patient was treated with CHOP and radiotherapy, and she is alive without evidence of disease after a 30-month follow-up. The diagnosis of an ALK-negative ALCL in an axillary lymph node of a patient with ipsilateral breast prosthesis and negative staging should prompt removal of the implant with capsulectomy, since the pathological study of this specimen allows the correct diagnosis with important prognostic implications.

Comparison of referring and final pathology for patients with T‐cell lymphoma in the National Comprehensive Cancer Network

T-cell lymphomas (TCLs) are uncommon in the United States. The accurate diagnosis of TCL is challenging and requires morphologic interpretation, immunophenotyping, and molecular techniques. The authors compared pathologic diagnoses at referring centers with diagnoses from expert hematopathology review to determine concordance rates and to characterize the usefulness of second-opinion pathology review for TCL. Patients in the National Comprehensive Cancer Network non-Hodgkin lymphoma database with peripheral TCL, not otherwise specified (PTCL-NOS), angioimmunoblastic TCL (AITL), and anaplastic lymphoma kinase (ALK)-positive and ALK-negative anaplastic large cell lymphoma (ALCL) were eligible if they had prior tissue specimens examined at a referring institution. Pathologic concordance was evaluated using available pathology and diagnostic testing reports and provider progress notes. The etiology of discordance and the potential impact on treatment were examined. Among 131 eligible patients, 57 (44%) had concordant results, totaling 64% of the 89 patients who were referred with a final diagnosis. Thirty-two patients (24%) had discordant results, representing 36% of those who were referred with a final diagnosis. The rates of discordance among patients with of PTCL-NOS, AITL, ALK-negative ALCL, and ALK-positive ALCL were 19%, 33%, 34%, and 6%, respectively. In 14 patients (44% of discordant results), pathologic reclassification could have resulted in a different therapeutic strategy. Forty-two patients (32%) were referred for classification with a provisional diagnosis. In a large cohort of patients with TCL who were referred to National Comprehensive Cancer Network centers, the likelihood of a concordant final diagnosis at a referring institution was low. As current and future therapies target TCL subsets, these data suggest that patients with suspected TCLs would benefit from evaluation by an expert hematopathologist.

CHOEP-21 Chemotherapy without Autologous Stem Cell Transplantation (ASCT) for Newly Diagnosed Nodal Peripheral T-Cell Lymphomas (PTCLs) in the Northern Part of Thailand

AbstractAbstract 4857 Background:Several previous studies demonstrated the poor treatment outcomes for the patients with nodal peripheral T-cell lymphomas (PTCLs) including PTCL, not otherwise specified (PTCL-NOS), ALK-negative anaplastic large cell lymphoma (ALCL) and angioimmunoblastic T-cell lymphoma (AITL) when compared with ALK-positive ALCL and aggressive B-cell lymphomas. For the treatment of T-cell lymphomas, promising results of chemotherapy regimens including etoposide in several studies were evidenced. We conducted the prospective study to determine the effectiveness and tolerability of the combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) with addition of etoposide (CHOEP-21) for newly diagnosed nodal peripheral T-cell lymphomas (PTCLs) treated at Maharaj NakornChiang Mai Hospital in the Northern part of Thailand. Method:From October 2008 to November 2011, patients ages 18–60 years with newly diagnosed nodal PTCLs excluding ALK-positive ALCL were enrolled to receive CHOEP-21 (CHOP plus etoposide 100 mg/m2 days 1–3) every 3 weeks for 8 cycles with granulocyte colony-stimulating factor (G-CSF) starting from day 4–11. None of them proceeded to undergo autologous stem cell transplantation (ASCT) due to financial problems. Event-free survival (EFS) which is the primary endpoint, was calculated from the date of diagnosis until disease progression or relapsed, change to other treatment, or death from any cause. Secondary end point of the study was overall survival (OS), response rate and toxicities of regimen. Results:24 patients were enrolled. Twenty of them were male with a mean age of 49 years. Majority of the patients had PTCL, NOS (n = 16), 7 patients had AITL and only one ALK-negative ALCL patient. Almost all except one were in stage III-IV with 62% of the patients had international prognostic index (IPI) ≥ 2. From median follow up of 16 months, overall response (OR) was 58% (33% CR) but 28.5% of these patients eventually relapsed as shown in table 1. Estimated 2-year EFS and OS were 33.8% and 55%, respectively. When compared with the 11 historical control receiving CHOP regimen, the outcomes including EFS, OS and OR were not statistically different, even though patients receiving CHOEP had more advanced stage and bone marrow involvement than those receiving CHOP regimen (Table 2, Figure 1 and 2). Infection (grade 3–4 of 8.3%) and hematologic toxicities were the most common adverse effects with acceptable grade 3–4 neutropenia (7.6%) and thrombocytopenia (4.6%). Conclusion:The CHOEP-21 without following ASCT did not seem to improve outcomes in patients with nodal PTCLs. Other treatment strategies are warranted to study in these groups of patients.Table 1Response to treatmentHistopathologyTotalCR (%)PR (%)PD (%)Relapsed (%)Alive (%)Alive in CR (%)PTCL, NOS163 (19)4 (17)9 (56)2 (13)9 (56)2 (13)AITL74 (57)2 (29)1 (14)2 (29)5 (71)3 (43)ALCL, ALK-negative11 (100)0 (0)0 (0)0 (0)1 (100)1 (100)Total248 (33)6 (25)10 (42)4 (17)15 (63)6 (25)Table 2Comparison between CHOEP and CHOP historical control patient characteristics and outcomeParameterCHOPCHOEPP valueNumber of patients1124-Age, mean+SD y (range)48.4+10.3 (25–60)48.9+9.9 (18–60)0.683Sex (male:female ratio)5: 65: 10.008Histologic subtype, no. patients (%)- PTCL, NOS7 (64)16 (64)0.666- ALCL, ALK-negative0 (0)1 (9)- AITL4 (36)7 (27)Ann Arbor stage III - IV, no. patients (%)5 (45)23 (95)0.006Bone marrow involvement, no. patients (%)1 (9)4 (36)0.017Elevated LDH serum level, no. patients (%)7 (64)14 (58)0.539IPI score, no. patients (%)0–16 (54)9 (38)0.022–33 (27)12 (50)4–52 (18)3 (12)CR (%)8 (72)8 (33)0.466Overall response rate; CR+PR (%)8 (72)14 (58)0.081Relapse/refractory (%)3 (27)10 (42)0.032 [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Leukemic Presentation of ALK-negative Anaplastic Large Cell Lymphoma in a Patient with Myelodysplastic Syndrome

A 50-year-old woman with a history of aplastic anemia developed cervical lymphadenopathy and atypical lymphocytosis. Atypical cells of lymph nodes were positive for CD3 and CD30 but negative for anaplastic lymphoma kinase (ALK). Bone marrow examination showed trilineage myelodysplasia. She was diagnosed with ALK-negative anaplastic large cell lymphoma (ALCL) with leukemic transformation and myelodysplastic syndrome (MDS) which presumably developed from aplastic anemia. The lymphoma was resistant to intensive chemotherapies, ultimately leading to death. Leukemic presentation of ALK-negative ALCL as an initial manifestation is extremely rare, and the progression of the disease may be influenced by MDS through alteration of immune functions.

Preclinical and Clinical Binding Properties, Internalization Kinetics, and Clinicopathological Activity of Brentuximab Vedotin (SGN-35): A Novel Antibody Drug Conjugate for Anaplastic Large Cell Lymphoma and Classical Hodgkin Lymphoma

AbstractAbstract 1789 Background:Brentuximab vedotin (SGN-35) is a novel anti-CD30 antibody conjugated to the cytotoxic drug monomethyl auristatin E (MMAE) designed to selectively target and kill CD30 expressing neoplasms. This agent has demonstrated antitumor activity in classical Hodgkin lymphoma and CD30-positive T cell lymphoma, yet the binding properties, internalization kinetics, and clinicopathological findings have not been described in tumor specimens derived from treated patients. Therefore, we investigated the activity of SGN-35 on a patient with cutaneous manifestations of systemic ALK-negative anaplastic large cell lymphoma (ALCL), and correlated these results with studies of the activity of SGN-35 on cultured CD30-positive cells lines. Methods and Results:First, we confirmed (using flow cytometry) that SGN-35 and the anti-CD30 antibody clone BerH83 do not compete for binding to CD30. Next, in Karpas 299 (ALCL cell line) and KM-H2 (cell line derived from Hodgkin lymphoma) cells, we quantitatively monitored CD30 expression and cell-surface SGN-35 over time (measured as antigen binding capacity [ABC]/cell), demonstrating maximum antigen expression at 24–48 hours of incubation with 15 μg/ml SGN-35 followed by decrease at 120 hours, consistent with SGN-35-induced internalization (Table). Similar studies were performed on sequential tumor biopsies from a 68 year old male with a 5-year history of ALK-negative ALCL relapsing after 4 prior systemic regimens and radiation, now presenting with multiple pink ulcerated tumorous lesions on his lower extremities as well as bone and nodal involvement. H&E and anti-CD30 immunohistochemical stains of both pretreatment and 24 hrs post-treatment (after the first dose of SGN-35 (1.8mg/kg)-ClinicalTrials.gov identifier NCT01026415) skin punch biopsies showed a dense dermal infiltrate of large CD30-positive neoplastic cells. Biopsy at 48 hrs post-treatment demonstrated numerous apoptotic cells. Antigen density experiments on the patient biopsy specimens and clinical findings correlated with these morphologic results. CD30 antigen density was highest on the patient's cells pretreatment biopsy (1.01 × 105 ABC units/cell) and then decreased after 24 hrs (7.83 × 104 ABC units/cell) and 48 hrs (5.08 × 104 ABC units/cell). Bound SGN-35 was greatest at 24 hrs (2.26 × 103 ABC units/cell) and decreased at 48 hrs (1.40 × 103 ABC units/cell) (Table). The corresponding measured blood concentrations of SGN-35 at 24 hr, 48 hr, and 21 days were 10 μg/mL, 7 μg/mL, and 1 μg/mL, respectively. No CD30 positive cells were present in the day 21 biopsy, precluding evaluation of CD30 antigen density and bound SGN-35. Clinically, the size and prominence of the skin lesions were reduced at day 21 post treatment and biopsy at this time point showed no morphologic or immunohistochemical evidence of the neoplastic population (pathologic complete remission). After the second dose of SGN-35 the patient achieved an 81% reduction in all radiographically measurable lesions (partial response) and achieved a radiographic and cutaneous complete remission after the 5th infusion of this agent. Conclusion:These data are the first to suggest in both cell lines and patient-derived tissues that the internalization kinetics of SGN-35 is rapid with resultant reduction in CD30 expression within the first 48 hours and concurrent apoptosis induction within the targeted cells. While the measured SGN-35 occupancy of CD30 binding sites in the patient is lower than with the cell lines, these data highlight the potential antitumor activity of this agent. The results from the single patient need to be confirmed in a cohort of patients. Nevertheless, such results imply that even with subsaturating occupancy of CD30, thousands of molecules of SGN-35 (each with approximately 4 molecules of MMAE) are likely to be internalized by and potentially kill each targeted cell and may be sufficient to yield pathologic remissions and clinical activity. Disclosures:Fromm:Seattle Genetics: Research Funding. McEarchern:Seattle Genetics: Employment. Kennedy:Seattle Genetics: Employment. Shustov:Seattle Genetics: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Gopal:Seattle Genetics: Research Funding.

Linkage of expression of chemokine receptors (CXCR3 and CCR4) and cytotoxic molecules in peripheral T cell lymphoma, not otherwise specified and ALK-negative anaplastic large cell lymphoma

Peripheral T cell lymphoma not otherwise specified (PTCL-N) and ALK-negative anaplastic large cell lymphoma (ALCL) are heterogeneous categories with poor diagnostic reproducibility. To clarify the biologic features of these categories, we investigated the expression of two chemokine receptors, type 1 (Th1/Tc1)-associated CXCR3 and type 2 (Th2/Tc2)-associated CCR4 in 110 PTCL-N and 35 ALK-negative ALCL cases, as well as the expression of cytotoxic molecules (CM). CXCR3 and CCR4 were expressed in 69 (63%) and 37 (34%) of PTCL-N, and in 12 (34%) and 6 (17%) of ALK-negative ALCL, respectively. In PTCL-N, type 1 pattern (CXCR3(+)CCR4(-)) was dominant (52%), whereas in ALK-negative ALCL, 54% were negative for both (P < 0.0001). CM was expressed in 38% of PTCL-N and 51% of ALK-negative ALCL. CM-positive PTCL-N consisted mostly of type 1 disease, which shows type 1 phenotype. In contrast, type 2 pattern (CXCR3(-)CCR4(+)) was recognized in the CM-negative group only. Among type 1 disease, CM-positive cases had a higher female ratio and more aggressive clinical features than CM-negative cases and a poorer prognosis (P = 0.006). Multivariate analysis confirmed that the type 1 phenotype with CM expression was an independent prognostic factor. In both PTCL-N and ALK-negative ALCL, CM-positive type 1 disease had an extremely poor prognosis.

Cytotoxic Molecule (CM)-Positive Classical Hodgkin Lymphoma: a Clinicopathologic Study in Comparison with Nodal Peripheral T-Cell Lymphoma of Not Otherwise Specified Type Possessing CM Expression.

Abstract 1549Poster Board I-572Classical Hodgkin lymphoma (CHL) is characterized by Hodgkin and Reed Sternberg (H-RS) cells, which are B-cell origin in many cases. Recently, we highlighted an adverse prognostic significance of cytotoxic molecule (CM) expression among CHL patients (Asano N, et al. J Clin Oncol. 2006). However, the clinical characteristics of CM-positive CHL still remain controversial. We here document the clinicopathologic profiles of 35 patients with CM-positive CHL, consisting of 23 men and 12 women with a median age of 50 years (range, 16 - 84 years). All patients had lymphoadenopathy, and 14 cases showed mediastinal involvement at presentation. Physical findings included hepatomegaly and splenomegaly in six and five patients, respectively. Four patients had a bulky mass, and nine showed stage IV disease. As for laboratory data, four patients had elevated white blood cell counts (greater than 15.0 × 103/mm3). Anemia (hemoglobin level; less than 10.5 g/dl) was also present in six patients. The pathological diagnoses were nodular sclerosis type (NS) in 22 cases and mixed cellularity (MC) in 12 cases. The H-RS cells of CM-positive CHL had a prototypic immunophenotype of CD15+ CD30+ and Fascin+. All of the cases completely lacked CD20 positivity on H-RS cells, while the expression of CD3e and CD45RO was found in 2 and 1, respectively. The H-RS cells were further positive for EBV RNA transcripts in 14 of 32 (44%) cases studied by in situ hybridization method. All except three cases were negative for Pax5. A clonal TCR-g chain gene arrangement was undetected in any of the cases with successful amplification of control GAPDH DNA by PCR analysis. No cases show B-cell clonality. 27 of 35 patients received systemic multi-agent chemotherapy consisting of first-line treatment regimens as follows: doxorubicin, bleomycin, vinblastine, and dacarbacin (ABVD) (22 patients); cyclophosphamide, doxorubicin, vincristin, and prednisone (CHOP) (2 patients); two with C-MOPP; and one BEACOPP regimens. Three patients died within 6 months, before completing induction treatment because of disease progression. Overall, 22 patients responded to first-line treatment: 13 with complete response and 9 with partial response. 13 of them had relapses, and 8 died with a clinical course ranging from 7 to 142 months. Effective therapeutic approaches should be explored for CM-positive CHL patients, who resist standard treatment for CHL. Comparing CM-positive nodal peripheral T-cell lymphomas of not otherwise specified type (PTCL-N)(n=55) with CM-positive CHL(n=35), no significant differences were detected in clinical parameters except for the frequency of elevated lactate dehydrogenase level in CM-positive PTCL-N. Immunophenotypically, CM-positive CHL cases showed significantly higher rates for CD15, CD30, and Fascin expression, while CM-positive PTCL-N cases showed significantly higher CD3e, CD8, CD45RO, and CXCR3 positivity. The survival curve of CM-positive CHL showed poorer prognosis than that of CM-negative CHL (P = .0002) and better than that of CM-positive PTCL-N (P = .002) (Figure). These findings suggest that CM-positive CHL is characterized by an unfavorable clinical feature, although their histologic and phenotypic features are more suggestive of CHL. Moreover, one case in CM-positive CHL had presented the skin lesion as a CD30-positive lymphoproliferative disorder or an ALK-negative anaplastic large cell lymphoma (ALCL) at the time of recurrence. These findings suggest that CM-positive CHL represent a distinct variant form based on clinicopathologic and phenotypic traits beyond the framework of CHL. Significant overlaps in biologic and morphologic features has been identified in CHL and non-Hodgkin lymphoma. CM-positive CHL cases may be ‘intermediate lymphoma‘ between CHL and PTCL or ALK-negative ALCL. Further studies, including array CGH profiling analysis are needed to clarify the origin of CM-positive CHL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Gene Expression Profile of Nodal T-Cell Lymphomas Identifies a Molecular Link between Angioimmunoblastic T-Cell Lymphoma (AITL) and Follicular Helper T Cells (TFH), and between CD30+ Peripheral T-Cell Lymphoma and ALK-Negative Anaplastic Large Cell Lymphoma (ALCL).

AITL and PTCL-U, the two most common forms of T-cell lymphomas in western countries, usually present as nodal disease and pursue an aggressive clinical course. AITL is commonly associated with a constellation of clinical symptoms and distinct pathological features. Conversely, PTCL-U lacks precise diagnostic criteria, and by default comprises cases not fulfilling criteria for other entities, including tumors with borderline features to ALCL and AITL. The genetic alterations and pathogenic mechanisms underlying AITL and PTCL-U are largely unknown. To determine whether the molecular signature of AITL and PTCL-U could help in distinguishing both entities and in understanding ther ontogeny, we performed gene expression profile (GEP) analysis of 15 PTCL-U tissue samples (6 CD30+ and 9 CD30−) and 19 AITL samples (including 2 sorted tumor cell suspensions) using Affymetrix HG-U133A Plus2.0 pan-genomic oligonucleotide microarrays, with comparison to that of previously published normal T-cell subsets (J Immunol 173:68; J Immunol 175: 7837; Blood 104: 1952). Principle component analysis (PCA, accumulated variance 95%) of all 33 tissue samples yielded three groups of tumors: one group of 12 AITLs, one group of 10 PTCLs-U and one mixed group comprising 5 AITLs (some with features borderline to PTCL-U) and 6 PTCLs-U (including 5 of 6 CD30+ tumors). The AITL molecular signature consisted of 442 genes with increased levels of expression in AITL compared to PTCL-U (t test, p<0.002), including genes encoding cell adhesion molecules, immune receptors, extracellular matrix components and several chemokines, B-cell-related and follicular dendritic cell-related genes, genes involved in endothelial and vascular biology, and several genes reported to belong to the gene expression signature of normal TFH cells (CXCL13, BCL6, PDCD1, CD40L, CD200). To specifically address the question of a molecular link beween AITL and TFH cells, we performed gene set enrichment analysis (GSEA) of our dataset using published gene sets specific of distinct normal T-cell subsets (TFH, TH1, TH2). Compared to that of PTCL-U, the molecular signature of AITL was significantly enriched in TFH-specific genes, and the enrichment was even higher for sorted AITL cells compared to AITL tissues. GSEA failed to disclose a molecular link between PTCL-U and known T-cell subsets (TH1, TH2, TFH). Compared to CD30− PTCL-U, CD30+ PTCL-U had lower expression of genes involved in TCR signalling (t test, p<0.002), and showed molecular similarities with ALK-negative ALCL. In conclusion, GEP of non-anaplastic nodal PTCL (1) segregates AITL and PTCL-U, supporting the basis for histotyping; (2) shows molecular analogies between AITL and TFH cells, strongly supporting the hypothesis of a histogenetic link; (3) suggests molecular analogies between CD30+ PTCL-U and ALK-negative ALCL.

Non‐mediastinal grey zone lymphomas and report from the workshop

Grey zone lymphomas represent borderline lesions between classical Hodgkin lymphoma (cHL) and other morphologically and immunophenotypically related diseases and entities like nodular lymphocyte predominant HL, T-cell rich B-cell lymphoma, ALK-negative anaplastic large cell lymphoma (ALCL), diffuse large B cell lymphoma (DLBCL) anaplastic variant and primary mediastinal LBCL. The sharp definition of morphological, immunophenotypical and molecular features of the "text-book cases" of each disease and the comparison with grey zone cases has reduced most of the latter cases. Two reports in this workshop dealt with the problematic non-mediastinal grey zone lymphomas, one with a cHL of T cell-type presenting in the skin as a ALK-negative ALCL and the other with the grey zone between cHL of B-cell type and ALK-negative ALCL.