STAT3 couples activated tyrosine kinase signaling to the oncogenic core transcriptional regulatory circuitry of anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is an aggressive, CD30+ T cell lymphoma of children and adults. ALK fusion transcripts or mutations in the JAK-STAT pathway are observed in most ALCL tumors, but the mechanisms underlying tumorigenesis are not fully understood. Here, we show that dysregulated STAT3 in ALCL cooccupies enhancers with master transcription factors BATF3, IRF4, and IKZF1 to form a core regulatory circuit that establishes and maintains the malignant cell state in ALCL. Critical downstream targets of this network in ALCL cells include the protooncogene MYC, which requires active STAT3 to facilitate high levels of MYC transcription. The core autoregulatory transcriptional circuitry activity is reinforced by MYC binding to the enhancer regions associated with STAT3 and each of the core regulatory transcription factors. Thus, activation of STAT3 provides the crucial link between aberrant tyrosine kinase signaling and the core transcriptional machinery that drives tumorigenesis and creates therapeutic vulnerabilities in ALCL.

Molecular Screening in Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma: Anaplastic Lymphoma Kinase Analysis, Next-Generation Sequencing Fusion Gene Detection, and T-Cell Receptor Immunoprofiling.

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) originates from the T-lineage and is marked by rearrangements of the ALK gene. More than 10 fusion partners with the ALK gene are known, with the most common being the t(2;5)(p23;q35) translocation resulting in the NPM1::ALK fusion. In 10% to 20% of the ALK+ ALCL cases, the ALK gene fuses with various other partners. Modern molecular techniques, especially next-generation sequencing (NGS), have eased the identification of ALK gene fusion partners and have allowed in-depth characterization of the T-cell receptor (TCR) repertoire. We devised a real-time quantitative reverse-transcription polymerase chain reaction to measure the expression of the translocated portion of the ALK gene. Fusion partners for the ALK gene were analyzed using rapid amplification of 5'cDNA ends (RACE) method or NGS. TCR immunoprofiling was performed by amplicon NGS. We studied 96 ALK+ ALCL patients. NPM1::ALK fusion gene was observed in 71 patients, ATIC::ALK in 9, and TPM3::ALK in 3. CLTC::ALK, MYH9::ALK, and RNF213::ALK fusions were identified in 2 patients each. We also discovered the TPM4::ALK and SATB1::ALK fusion genes, plus the following 2 previously unidentified ALK+ ALCL fusions: SQSTM1::ALK and CAPRIN1::ALK. High expression of the translocated ALK gene segment was observed in all 93 analyzed samples. TCR testing was conducted on 23 patients with available DNA. In 18 (78%) patients, we discerned at least one (ranging from 1 to 4) clonal TCR rearrangement. In 59% of the patients, clonal TCR beta junctions corresponded with sequences previously observed in both healthy donors and under various pathological conditions. Reverse-transcriptase quantitative detection of ALK expression is a fast and reliable method for both diagnosing and monitoring treatment response in ALK+ ALCL patients, irrespective of the ALK gene translocation. NGS reveals new ALK translocation partners. Both malignant and reactive TCR repertoires in ALK+ ALCL patients are unique and do not consistently occur among different patients.

Extranodal NK/T-Cell Lymphoma Predominantly Composed of Anaplastic Cells: A Frequently Misdiagnosed and Highly Aggressive Variant.

Extranodal NK/T-cell lymphoma (ENKTL) is a non-Hodgkin lymphoma associated with the Epstein-Barr virus that primarily affects individuals in East Asia and indigenous populations in Central and South America. Morphologically, ENKTL typically consists of medium-sized cells or a combination of small and large cells. This report presents 10 cases characterized by predominantly anaplastic cells with diffuse expression of CD30, resembling anaplastic lymphoma kinase-negative anaplastic large cell lymphoma (ALK-negative ALCL) and demonstrating highly aggressive behavior. The cohort included 9 males and 1 female, ranging in age from 29 to 65 years (median age: 47 y). Eight patients presented with nasal disease, while 2 had non-nasal disease. Five patients had stage I/II disease, and the remaining 5 had stage III/IV disease. Morphologically, necrosis was observed in 9 cases, angiocentric-angiodestructive growth in 3 cases, and pseudoepitheliomatous hyperplasia in 2 cases. Anaplastic cells predominated in all cases, with some displaying eccentric, horseshoe-shaped, or kidney-shaped nuclei (referred to as "Hallmark" cells). The morphology profile was monomorphic in 3 cases and polymorphic in 7 cases. Immunohistochemically, all cases tested positive for cytotoxic granule markers (TIA1 and granzymeB) and Epstein-Barr virus-encoded RNA. Cytoplasmic expression of CD3ε and CD56 was observed in 9 of 10 cases. Interestingly, most cases (7 of 8) exhibited variable expression of MuM1, ranging from 10% to 90%. All cases showed diffuse positivity for CD30 but were negative for ALK, resulting in 3 cases being initially misdiagnosed as ALK-negative ALCL. Compared with nonanaplastic cases, anaplastic cells predominant ENKTL had a significantly higher frequency of "B" symptoms, bone marrow involvement, hemophagocytic lymphohistiocytosis, and higher Ki67 proliferative index. These findings provide valuable information for pathologists, expanding their understanding of the cytologic spectrum of ENKTL. This rare variant of ENKTL, characterized by the predominance of anaplastic cells and diffuse CD30 expression, exhibits high aggressiveness and should be differentiated from ALK-negative ALCL. Awareness of this uncommon variant is crucial in preventing misdiagnosis and ensuring the timely initiation of therapy.

Detection of TRAF1-ALK fusion in skin lesions of systemic ALK+ anaplastic large cell lymphoma initially involving the skin and the draining lymph node.

A case of cytoplasmic anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) initially involving the skin in a 44-year-old Japanese female is reported. The patient had a hemorrhagic erythematous tumor on the right thigh without any systemic symptoms. Pathology showed diffuse infiltration of CD30-positive anaplastic large cells positive for epithelial membrane antigen and cytoplasmic ALK. The right inguinal lymph node showed infiltration of tumor cells in the marginal sinus. Only 2 weeks after radiation therapy, the patient developed multiple subcutaneous nodules and lung involvement. Even after subsequent multichemotherapy sessions, cutaneous recurrence occurred. Literature review of cytoplasmic ALK-positive ALCL initially involving in the skin revealed that skin lesions were mostly seen in the extremities and that half of the cases developed extracutaneous lesions. Radiation and chemotherapy were effective for most cases. Inverse RT-PCR identified a tumor necrosis factor receptor-associated factor (TRAF)1-ALK fusion in our case. Most reported cases with this translocation experienced repeated changes in chemotherapy, suggesting poorer prognosis. Although ALK-positive ALCL generally responds well to chemotherapy, the presence of a TRAF1-ALK fusion may suggest resistance to treatment. Detection of fusion partners of ALK is important for predicting clinical courses and deciding treatment options.

Spatially Resolved Transcriptomes of CD30+-Transformed Mycosis Fungoides and Cutaneous Anaplastic Large-Cell Lymphoma.

Mycosis fungoides with large-cell transformation (MF-LCT) occurs in a minor proportion of aggressive lesions, which express CD30 similar to primary cutaneous anaplastic large-cell lymphoma (pcALCL). We investigated the differences in spatially resolved transcriptome profiles of MF-LCT and pcALCL using CD30 morphology markers and 28 and 24 regions of interest (ROIs) in MF-LCT and pcALCL, respectively. Differentially expressed genes, pathway analysis, and immune-cell deconvolution by selective analysis of CD30-positive tumor cells and CD30-negative extratumoral areas were undertaken. In CD30-positive ROIs of MF-LCT, 190 differentially expressed genes were upregulated (29 were directly or indirectly associated with extracellular matrix remodeling), whereas 255 differentially expressed genes were downregulated, compared with those of pcALCL. Except for cornified envelope formation and keratinization, all six pathways enriched in CD30-positive ROIs of MF-LCT were associated with extracellular matrix remodeling. In CD30-positive ROIs in MF-LCT compared with those in pcALCL, immune-cell deconvolution revealed significantly increased fibroblasts and M2 macrophages (P = 0.012 and P = 0.023, respectively) but decreased M1 macrophages (P = 0.031). In CD30-negative ROIs in MF-LCT compared with those in pcALCL, memory B (P = 0.021), plasma (P = 0.023), and CD8 memory T (P = 0.001) cells significantly decreased, whereas regulatory T cells (P = 0.024) increased. Predomination of extracellular matrix remodeling pathways and immunosuppressive microenvironment in MF-LCT indicates pathophysiological differences between MF-LCT and pcALCL.

Pathobiology of nodal peripheral T-cell lymphomas: current understanding and future directions.

Predominantly nodal is the most common clinical presentation of peripheral T- (and NK-) cell lymphomas (PTCL), which comprise three main groups of diseases: (i) systemic anaplastic large cell lymphomas (ALCL), whether positive or negative for anaplastic lymphoma kinase (ALK); (ii) follicular helper T-cell lymphomas (TFHL); and (iii) PTCL, not otherwise specified (NOS). Recent advances in the genomic and molecular characterization of PTCL, with enhanced understanding of pathobiology, have translated into significant updates in the latest 2022 classifications of lymphomas. ALK-negative ALCL is now recognized to be genetically heterogeneous, with identification of DUSP22 rearrangements in approximately 20-30% of cases, correlated with distinctive pathological and biological features. The notion of cell-of-origin as an important determinant of the classification of nodal PTCL is best exemplified by TFHL, considered as one disease or a group of related entities, sharing oncogenic pathways with frequent recurrent epigenetic mutations as well as a relationship to clonal hematopoiesis. Data are emerging to support that a similar cell-of-origin concept might be relevant to characterize meaningful subgroups within PTCL, NOS, based on cytotoxic and/or Th1 versus Th2 signatures. The small group of primary nodal Epstein-Barr virus-positive lymphomas of T- or NK-cell derivation, formerly considered PTCL, NOS, is now classified separately, due to distinctive features, and notably an aggressive course. This review summarizes current knowledge of the pathology and biology of nodal-based PTCL entities, with an emphasis on recent findings and underlying oncogenic mechanisms.

Differential molecular programs of cutaneous anaplastic large cell lymphoma and CD30-positive transformed mycosis fungoides.

Background: Discriminating between cutaneous anaplastic large cell lymphoma (cALCL) and CD30-positive transformed mycosis fungoides (CD30+ TMF) is challenging, particularly when they arise in the context of pre-existing mycosis fungoides. The development of molecular diagnostic tools was hampered by the rarity of both diseases and the limited understanding of their pathogenesis. Methods: In this study, we established a cohort comprising 25 cALCL cases and 25 CD30+ TMF cases, with transcriptomic data obtained from 31 samples. We compared the clinicopathological information and investigated the gene expression profiling between these two entities. Furthermore, we developed an immunohistochemistry (IHC) algorithm to differentiate these two entities clinically. Results: Our investigation revealed distinct clinicopathological features and unique gene expression programs associated with cALCL and CD30+ TMF. cALCL and CD30+ TMF displayed marked differences in gene expression patterns. Notably, CD30+ TMF demonstrated enrichment of T cell receptor signaling pathways and an exhausted T cell phenotype, accompanied by infiltration of B cells, dendritic cells, and neurons. In contrast, cALCL cells expressed high levels of HLA class II genes, polarized towards a Th17 phenotype, and exhibited neutrophil infiltration. An IHC algorithm with BATF3 and TCF7 staining emerged as potential diagnostic markers for identifying these two entities. Conclusions: Our findings provide valuable insights into the differential molecular signatures associated with cALCL and CD30+ TMF, which contribute to their distinct clinicopathological behaviors. An appropriate IHC algorithm could be used as a potential diagnostic tool.

Chimeric kinase ALK induces expression of NAMPT and selectively depends on this metabolic enzyme to sustain its own oncogenic function.

As we show in this study, NAMPT, the key rate-limiting enzyme in the salvage pathway, one of the three known pathways involved in NAD synthesis, is selectively over-expressed in anaplastic T-cell lymphoma carrying oncogenic kinase NPM1::ALK (ALK + ALCL). NPM1::ALK induces expression of the NAMPT-encoding gene with STAT3 acting as transcriptional activator of the gene. Inhibition of NAMPT affects ALK + ALCL cells expression of numerous genes, many from the cell-signaling, metabolic, and apoptotic pathways. NAMPT inhibition also functionally impairs the key metabolic and signaling pathways, strikingly including enzymatic activity and, hence, oncogenic function of NPM1::ALK itself. Consequently, NAMPT inhibition induces cell death in vitro and suppresses ALK + ALCL tumor growth in vivo. These results indicate that NAMPT is a novel therapeutic target in ALK + ALCL and, possibly, other similar malignancies. Targeting metabolic pathways selectively activated by oncogenic kinases to which malignant cells become "addicted" may become a novel therapeutic approach to cancer, alternative or, more likely, complementary to direct inhibition of the kinase enzymatic domain. This potential therapy to simultaneously inhibit and metabolically "starve" oncogenic kinases may not only lead to higher response rates but also delay, or even prevent, development of drug resistance, frequently seen when kinase inhibitors are used as single agents.

Nuclear NPM-ALK Protects Myc from Proteasomal Degradation and Contributes to Its High Expression in Cancer Stem-Like Cells in ALK-Positive Anaplastic Large Cell Lymphoma.

In ALK-positive anaplastic large cell lymphoma (ALK+ALCL), a small subset of cancer stem-like (or RR) cells characterized by high Myc expression have been identified. We hypothesize that NPM-ALK contributes to their high Myc expression. While transfection of NPM-ALK into HEK293 cells effectively increased Myc by inhibiting its proteosomal degradation (PD-Myc), this effect was dramatically attenuated when the full-length NPM1 (FL-NPM1) was downregulated using shRNA, highlighting the importance of the NPM-ALK:FL-ALK heterodimers in this context. Consistent with this concept, immunoprecipitation experiments showed that the heterodimers are abundant only in RR cells, in which the half-life of Myc is substantially longer than the bulk cells. Fbw7γ, a key player in PD-Myc, is sequestered by the heterodimers in RR cells, and this finding correlates with a Myc phosphorylation pattern indicative of ineffective PD-Myc. Using confocal microscopy and immunofluorescence staining, we found that the fusion signal between ALK and FL-NPM1, characteristic of the heterodimers, correlates with the Myc level in ALK+ALCL cells from cell lines and patient samples. To conclude, our findings have revealed a novel oncogenic function of NPM-ALK in the nucleus. Specifically, the NPM-ALK:FL-NPM1 heterodimers increase cancer stemness by blocking PD-Myc and promoting Myc accumulation in the cancer stem-like cell subset.

Anaplastic large cell lymphomas with equivocal DUSP22 FISH results: recommendations for clinical reporting and diagnostic evaluation.

Anaplastic large cell lymphoma (ALCL), one of the most common T-cell lymphomas, shows unifying pathological features but is clinically and genetically heterogeneous. One genetic subgroup, characterized by recurrent DUSP22 rearrangements (R), has distinct morphologic, immunophenotypic, and molecular features and can be identified in routine pathology practice using a breakapart (BAP) fluorescence in situ hybridization (FISH) probe. However, some cases show equivocal BAP-FISH findings (BAP-FISHEQ) and the features of these cases are poorly understood. Here, we sought to characterize DUSP22 BAP-FISHEQ ALCLs further. First, we applied an immunohistochemistry (IHC) algorithm using TIA1, pSTAT3Y705, and LEF1, which can predict DUSP22-R with high accuracy. Among 37 BAP-FISHEQ ALCLs, 18 (49%) were IHC-algorithm positive (IHCPOS), 8 (21%) were IHC-algorithm negative (IHCNEG), and 11 (30%) were IHCEQ. In 32 BAP-FISHEQ cases, we also applied a dual-color, dual-fusion (D-FISH) probe for t(6;7)(p25.3;q32.3), which accounts for 45% of DUSP22-R ALCLs. Among BAP-FISHEQ cases, D-FISH was positive in 10/18 IHCPOS cases (56%), 0/9 IHCEQ cases (0%), and 0/5 IHCNEG cases (0%). Median survival in BAP-FISHEQ ALCLs was 105 months, intermediate between BAP-FISHPOS ALCLs (median survival not reached) and BAP-FISHNEG ALCLs (19 months). Thus, DUSP22 BAP-FISHEQ ALCLs are clinicopathologically heterogeneous, likely due to an admixture of cases with an unbalanced DUSP22-R and cases with focal deletions without rearrangement. For clinical reporting, we recommend that DUSP22 BAP-FISHEQ ALCLs be reported as equivocal, and not be grouped with BAP-FISHPOS ALCLs. Clinical adoption of an IHC algorithm, possibly supplemented by t(6; 7) D-FISH, could facilitate genetic subtyping in about two-thirds of BAP-FISHEQ ALCLs.

MiR-146a-5p enrichment in small-extracellular vesicles of relapsed pediatric ALCL patients promotes macrophages infiltration and differentiation.

Anaplastic large cell lymphoma (ALCL) is a CD30-positive lymphoma accounting for 20% of all pediatric T-cell lymphomas. Current first line treatment can cure most of ALCL patients but 10-30% of them are resistant or relapse. In this context, liquid biopsy has the potential to help clinicians in disease screening and treatment response monitoring. Small-RNA-sequencing analysis performed on plasma small-extracellular vesicles (s-EVs) from 20 pediatric anaplastic lymphoma kinase positive (ALK + ) ALCL patients at diagnosis revealed a specific miRNAs cargo in relapsed patients compared to non-relapsed, with seven miRNAs enriched in s-EVs of relapsed patients. MiR-146a-5p and miR-378a-3p showed a negative prognostic impact both in univariate and multivariate analysis, possibly representing, together with let-7 g-5p, a miRNA panel for the early identification of high-risk patients. Among them, miR-146a-5p is known to modulate tumor supporting-M2 macrophages differentiation, but the role of these cells in pediatric ALK + ALCL is still unknown. To elucidate the role of miR-146a-5p and M2 macrophages in pediatric ALCL disease, THP-1-derived macrophages were treated with s-EVs from ALK + ALCL cell lines, showing increased miR-146a-5p intracellular expression, migrating capability and M2-markers CD163 and Arginase-1 upregulation. In turn, conditioned media from M2 macrophages or miR-146a-5p-transfected THP-1 increased ALCL cells' aggressive features and were enriched in interleukin-8. Overall, these data suggest a role of miR-146a-5p in promoting macrophage infiltration and M2-like polarization in ALCL. Our findings incite further investigation on the role of M2 macrophages in ALCL aggressiveness and dissemination, also considering the novel treatment options targeting tumor associated macrophages.

Prognostic factors for primary cutaneous anaplastic large-cell lymphoma: a multicentre retrospective study from Japan.

BACKGROUND: The clinical implications of DUSP22 rearrangement and the association between DUSP22 rearrangement and lymphoid enhancer-binding factor 1 (LEF1) expression pattern in CD30+ cutaneous T-cell lymphomas (CTCLs) are unknown. OBJECTIVES: This study assessed the incidence of DUSP22 rearrangement and its clinical and immunohistochemical implications in primary cutaneous anaplastic large-cell lymphoma (pcALCL), lymphomatoid papulosis (LyP) and CD30+ mycosis fungoides with large-cell transformation (MF-LCT), focusing especially on the association with the prognosis and LEF1 expression pattern. Prognostic factors of pcALCL were also examined. METHODS: We conducted a multicentre retrospective study including patients with pcALCL, LyP and MF-LCT diagnosed between 1 January 2000 and 31 December 2018 in Japan. Baseline data at diagnosis, treatment course, overall survival (OS) and disease-specific survival (DSS) were collected. Immunohistochemical analysis and fluorescence in situ hybridization to detect DUSP22 and TP63 rearrangement were performed using skin samples at diagnosis. We investigated the association between staining pattern and these gene rearrangements. We also assessed the prognostic implications of clinical status, immunohistochemical results and the presence of gene rearrangements. RESULTS: DUSP22 rearrangement was detected in 50% (11 of 22) of cases of pcALCL, but not in any cases with LyP (0 of 14) or MF-LCT (0 of 11). TP63 rearrangement was not detected in any case. Clinically, patients with pcALCL with DUSP22 rearrangement did not tend to develop ulcers (P = 0.081). There was no significant association between DUSP22 rearrangement status and immunohistochemical results, including LEF1 expression pattern. T3 stage and the presence of lower limb lesions were significantly associated with shorter OS (P = 0.012 and 0.021, respectively, by log-rank test). Similarly, they were significantly correlated with shorter DSS (P = 0.016 and 0.0001, respectively). CONCLUSIONS: DUSP22 rearrangement is relatively specific to pcALCL among CD30+ CTCLs in Japan. Although the LEF1 expression pattern was not related to DUSP22 rearrangement in pcALCL, there was no rearrangement if LEF1 was not expressed. We confirmed that T3 stage and the lower limb involvement were significantly associated with decreased OS and DSS. The presence or absence of lower limb lesions should be included in T-stage subcategorization in the future.

Targeting CCR7-PI3Kγ overcomes resistance to tyrosine kinase inhibitors in ALK-rearranged lymphoma.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Analysis and therapeutic targeting of the IL-1R pathway in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL), a subgroup of mature T-cell neoplasms with an aggressive clinical course, is characterized by elevated expression of CD30 and anaplastic cytology. To achieve a comprehensive understanding of the molecular characteristics of ALCL pathology and to identify therapeutic vulnerabilities, we applied genome-wide CRISPR library screenings to both anaplastic lymphoma kinase positive (ALK+) and primary cutaneous (pC) ALK- ALCLs and identified an unexpected role of the interleukin-1R (IL-1R) inflammatory pathway in supporting the viability of pC ALK- ALCL. Importantly, this pathway is activated by IL-1α in an autocrine manner, which is essential for the induction and maintenance of protumorigenic inflammatory responses in pC-ALCL cell lines and primary cases. Hyperactivation of the IL-1R pathway is promoted by the A20 loss-of-function mutation in the pC-ALCL lines we analyze and is regulated by the nonproteolytic protein ubiquitination network. Furthermore, the IL-1R pathway promotes JAK-STAT3 signaling activation in ALCLs lacking STAT3 gain-of-function mutation or ALK translocation and enhances the sensitivity of JAK inhibitors in these tumors in vitro and in vivo. Finally, the JAK2/IRAK1 dual inhibitor, pacritinib, exhibited strong activities against pC ALK- ALCL, where the IL-1R pathway is hyperactivated in the cell line and xenograft mouse model. Thus, our studies revealed critical insights into the essential roles of the IL-1R pathway in pC-ALCL and provided opportunities for developing novel therapeutic strategies.

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.

New perspectives for targeting therapy in ALK-positive human cancers.

Anaplastic lymphoma kinase (ALK) is a member of the insulin receptor protein-tyrosine kinase superfamily and was first discovered in anaplastic large-cell lymphoma (ALCL). ALK alterations, including fusions, over-expression and mutations, are highly associated with cancer initiation and progression. This kinase plays an important role in different cancers, from very rare to the more prevalent non-small cell lung cancers. Several ALK inhibitors have been developed and received Food and Drug Administration (FDA) approval. However, like other drugs used in targeted therapies, ALK inhibitors inevitably encounter cancer cell resistance. Therefore, monoclonal antibody screening based on extracellular domain or combination therapies may provide viable alternatives for treating ALK-positive tumors. In this review, we discuss the current understanding of wild-type ALK and fusion protein structures, the pathological functions of ALK, ALK target therapy, drug resistance and future therapeutic directions.

How to Diagnose Anaplastic Large Cell Lymphoma on Cytological Samples? A Series with Emphasis on Diagnostic Clue and Pitfalls.

INTRODUCTION: Anaplastic large cell lymphoma (ALCL) is a rare mature T-cell non-Hodgkin's lymphoma characterized by large and pleomorphic neoplastic CD30-positive T cells. ALCL includes different subtypes with different clinical and biological features: systemic ALCL, primary cutaneous ALCL, breast implant-associated ALCL (BIA-ALCL). Anaplastic lymphoma kinase (ALK) is overexpressed and rearranged in some systemic cases. Diagnosis of ALCL may be challenging on cytological samples, but the correct diagnosis is mandatory for the management of the patient. METHODS: A retrospective series of 12 ALCLs diagnosed by cytology is reported. Cytological samples included lymph nodes and skin lesions fine needle aspiration cytology, peritoneal effusion, and periprosthetic fluid. Microscopic evaluation was performed on direct smears, cell-block sections, and cytocentrifugated slides. Immunocytochemistry was performed on cell-block sections, direct smears, and cytocentrifugated slides. Molecular evaluation by fluorescent in-situ hybridization (FISH) was performed on cell-block sections. RESULTS: The series included 4 ALK+ ALCLs, 5 ALK- ALCLs, and 3 BIA-ALCLs. FNAC was performed on lymph nodes in 8 cases and on skin lesion in 1 case. In this last case, a peritoneal effusion was also evaluated. Breast periprosthetic fluids were evaluated in 3 cases. A large immunocytochemical panel was performed in each case, and FISH in 3 cases, demonstrating ALK rearrangement in a case of ALK+ ALCL. A final diagnosis was rendered in all cases. In the case of skin lesion, the differential diagnosis between systemic ALCL and primary cutaneous ALCL was possible. CONCLUSION: The cytological diagnosis of ALCL may be challenging, and the proper management of the collected sample is mandatory. The rapid on-site evaluation and the realization of a cell block are strongly recommended. Immunocytochemistry is mandatory for the diagnosis and a large antibodies panel is needed as differential diagnosis includes many different neoplasms. FISH may be useful to evaluate ALK rearrangements. When properly managed, cytology can lead to a reliable final diagnosis of ALCL.