Second Neoplasms in Italian Patients with Hairy Cell Leukemia after Treatment with Cladribine: A Multicenter Investigation and Literature Review.

Concern has emerged about the prevalence of second cancers among patients with hairy cell leukemia (HCL) treated with purine analogs. We investigated 513 patients with HCL treated with cladribine over the last 30 years at 18 Italian centers and calculated their standardized incidence ratios (SIRs). We identified 24 patients with a second cancer diagnosed at a median time from treatment with cladribine of 59.9 months (range: 9.2-169.7 months). All patients with solid neoplasms presented with a limited-stage disease, except four cases of locally advanced cancer; multiple myeloma patients had a smoldering disease, while lymphoma patients had stage Ie and stage IV diseases. Response to therapy was complete in 19 cases; 1 patient is still receiving treatment for a relapsing bladder disease, while 2 patients progressed during treatment and died. These two patients died from unrelated causes: one from infection and one due to surgery complications. The median OS from HCL was 98.5 months (range: 38.4-409.2 months), while the median OS from second cancer was 27.6 months (range: 1-117.8 months). The SIR was 0.86 (95% CI: 0.54-1.30) for males and 1.13 (95% CI: 0.36-2.73) for females: no statistically significant differences were highlighted. We were not able to demonstrate an excess of second cancer or a significant association with the specific studied neoplasm.

An unusual form of BRAF-V600E mutated hairy cell neoplasm with clinical resistance to BRAF inhibition: Challenging precision medicine.

Distinct outcomes of BRAF inhibition in BRAF-mutated hairy cell neoplasms with wild-type or mutant TP53, and alternative strategies to overcome mutant TP53.

B-cell receptor reactivity against Rothia mucilaginosa in nodular lymphocyte-predominant Hodgkin lymphoma.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a Hodgkin lymphoma expressing functional B-cell receptors (BCR). Recently, we described a dual stimulation model of IgD+ lymphocyte-predominant cells by Moraxella catarrhalis antigen RpoC and its superantigen MID/hag, associated with extralong CDR3 and HLA-DRB1*04 or HLADRB1* 07 haplotype. The aim of the present study was to extend the antigen screening to further bacteria and viruses. The fragment antibody-binding (Fab) regions of seven new and 15 previously reported cases were analyzed. The reactivity of non-Moraxella spp.-reactive Fab regions against lysates of Rothia mucilaginosa was observed in 5/22 (22.7%) cases. Galactofuranosyl transferase (Gltf) and 2,3-butanediol dehydrogenase (Bdh) of R. mucilaginosa were identified by comparative silver- and immuno-staining in two-dimensional gels, with subsequent mass spectrometry and validation by western blots and enzyme-linked immunosorbent assay. Both R. mucilaginosa Gltf and Bdh induced BCR pathway activation and proliferation in vitro. Apoptosis was induced by recombinant Gltf/ETA'-immunotoxin conjugates in DEV cells expressing recombinant R. mucilaginosa-reactive BCR. Reactivity against M. catarrhalis RpoC was confirmed in 3/7 newly expressed BCR (total 10/22 reactive to Moraxella spp.), resulting in 15/22 (68.2%) cases with BCR reactivity against defined bacterial antigens. These findings strengthen the hypothesis of bacterial trigger contributing to subsets of NLPHL.

Genomic landscape of virus-associated cancers.

It has been estimated that 15%-20% of human cancers are attributable to infections, mostly by carcinogenic viruses. The incidence varies worldwide, with a majority affecting developing countries. Here, we present a comparative analysis of virus-positive and virus-negative tumors in nine cancers linked to five viruses. We find that virus-positive tumors occur more frequently in males and show geographical disparities in incidence. Genomic analysis of 1,658 tumors reveals virus-positive tumors exhibit distinct mutation signatures and driver gene mutations and possess a lower somatic mutation burden compared to virus-negative tumors of the same cancer type. For example, compared to the respective virus-negative counterparts, virus-positive cases across different cancer histologies had less often mutations of TP53 and deletions of 9p21.3/ CDKN2 A- CDKN1A ; Epstein-Barr virus-positive (EBV+) gastric cancer had more frequent mutations of EIF4A1 and ARID1A and less marked mismatch repair deficiency signatures; and EBV-positive cHL had fewer somatic genetic lesions of JAK-STAT, NF-κB, PI3K-AKT and HLA-I genes and a less pronounced activity of the aberrant somatic hypermutation signature. In cHL, we also identify germline homozygosity in HLA class I as a potential risk factor for the development of EBV-positive Hodgkin lymphoma. Finally, an analysis of clinical trials of PD-(L)1 inhibitors in four virus-associated cancers suggested an association of viral infection with higher response rate in patients receiving such treatments, which was particularly evident in gastric cancer and head and neck squamous cell carcinoma. These results illustrate the epidemiological, genetic, prognostic, and therapeutic trends across virus-associated malignancies.

Overlapping features of therapy-related and de novo NPM1-mutated AML.

NPM 1-mutated acute myeloid leukemia (AML) shows unique features. However, the characteristics of "therapy-related" NPM1-mutated AML (t-NPM1 AML) are poorly understood. We compared the genetics, transcriptional profile, and clinical outcomes of t-NPM1 AML, de novo NPM1-mutated AML (dn-NPM1 AML), and therapy-related AML (t-AML) with wild-type NPM1 (t-AML). Normal karyotype was more frequent in t-NPM1 AML (n = 78/96, 88%) and dn-NPM1 (n = 1986/2394, 88%) than in t-AML (n = 103/390, 28%; P < .001). DNMT3A and TET2 were mutated in 43% and 40% of t-NPM1 AML (n = 107), similar to dn-NPM1 (n = 88, 48% and 30%; P > 0.1), but more frequently than t-AML (n = 162; 14% and 10%; P < 0.001). Often mutated in t-AML, TP53 and PPM1D were wild-type in 97% and 96% of t-NPM1 AML, respectively. t-NPM1 and dn-NPM1 AML were transcriptionally similar, (including HOX genes upregulation). At 62 months of median follow-up, the 3-year overall survival (OS) for t-NPM1 AML (n = 96), dn-NPM1 AML (n = 2394), and t-AML (n = 390) were 54%, 60%, and 31%, respectively. In multivariable analysis, OS was similar for the NPM1-mutated groups (hazard ratio [HR] 0.9; 95% confidence interval [CI], 0.65-1.25; P = .45), but better in t-NPM1 AML than in t-AML (HR, 1.86; 95% CI, 1.30-2.68; P < .001). Relapse-free survival was similar between t-NPM1 and dn-NPM1 AML (HR, 1.02; 95% CI, 0.72-1.467; P = .90), but significantly higher in t-NPM1 AML versus t-AML (HR, 1.77; 95% CI, 1.19-2.64; P = .0045). t-NPM1 and dn-NPM1 AML have overlapping features, suggesting that they should be classified as a single disease entity.

SARS-CoV-2 infection and vaccination in patients with hairy-cell leukaemia.

Little is known of the course of COVID-19 and the antibody response to infection or vaccination in patients with hairy-cell leukaemia (HCL). Among a total of 58 HCL cases we studied in these regards, 37 unvaccinated patients, mostly enjoying a relatively long period free from anti-leukaemic treatment, developed COVID-19 between March 2020 and December 2021 with a usually favourable outcome (fatality rate: 5/37, 14%); however, active leukaemia, older age and more comorbidities were associated with a worse course. Postinfection (n = 11 cases) and postvaccination (n = 28) seroconversion consistently developed, except after recent anti-CD20 or venetoclax therapy, correlating with perivaccine B-cell count. Vaccination appeared to protect from severe COVID-19 in 11 patients with breakthrough infection.

Consensus opinion from an international group of experts on measurable residual disease in hairy cell leukemia.

A significant body of literature has been generated related to the detection of measurable residual disease (MRD) at the time of achieving complete remission (CR) in patients with hairy cell leukemia (HCL). However, due to the indolent nature of the disease as well as reports suggesting long-term survival in patients treated with a single course of a nucleoside analog albeit without evidence of cure, the merits of detection of MRD and attempts to eradicate it have been debated. Studies utilizing novel strategies in the relapse setting have demonstrated the utility of achieving CR with undetectable MRD (uMRD) in prolonging the duration of remission. Several assays including immunohistochemical analysis of bone marrow specimens, multi-parameter flow cytometry and molecular assays to detect the mutant BRAF V600E gene or the consensus primer for the immunoglobulin heavy chain gene (IGH) rearrangement have been utilized with few comparative studies. Here we provide a consensus report on the available data, the potential merits of MRD assessment in the front-line and relapse settings and recommendations on future role of MRD assessment in HCL.

Development of a distributed international patient data registry for hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare lymphoproliferative disorder, comprising only 2% of all leukemias. The Hairy Cell Leukemia Foundation (HCLF) has developed a patient data registry to enable investigators to better study the clinical features, treatment outcomes, and complications of patients with HCL. This system utilizes a centralized registry architecture. Patients are enrolled at HCL Centers of Excellence (COE) or via a web-based portal. All data are de-identified, which reduces regulatory burden and increases opportunities for data access and re-use. To date, 579 patients have been enrolled in the registry. Efforts are underway to engage additional COE's to expand access to patients across the globe. This international PDR will enable researchers to study outcomes in HCL in ways not previously possible due to the rarity of the disease and will serve as a platform for future prospective research.

Long-term follow-up of cladribine treatment in hairy cell leukemia: 30-year experience in a multicentric Italian study.

Hairy cell leukemia (HCL) is a rare lymphoproliferative disease with an excellent prognosis after treatment with cladribine (2CDA), although relapse may occur during follow-up. The aim of the study is to review the efficacy, safety, long-term remission rate, and overall survival (OS) in those patients who received 2CDA as first-line treatment. We retrospectively reviewed data of HCL patients treated with 2CDA between March 1991 and May 2019 at 18 Italian Hematological centers: 513 patients were evaluable for study purpose. The median age was 54 years (range 24-88) and ECOG was 0 in 84.9% of cases. A total of 330 (64.3%) patients received 2CDA intravenously and 183 (35.7%) subcutaneously. ORR was 91.8%: CR was obtained in 335 patients (65.3%), PR in 96 (18.7%), and hematological response in 40 (7.8%) patients; in 42 (8.2%) no response was observed. Hemoglobin value (p = 0.044), frequency of circulating hairy cells (p = 0.039), recovery of absolute neutrophil count (p = 0.006), and normalization of spleen (p ≤ 0.001) were associated with CR compared to PR in univariable analysis. At a median follow-up of 6.83 years (range 0.04-28.52), the median time to relapse was 12.2 years. A significant difference in duration of response was identified between patients that obtained a CR and PR (19.4 years versus 4.8 years, p < 0.0001). Non-hematological grade 3 or higher early toxicity was reported in 103 (20.1%) patients. Median OS was not reached: 95.3%, 92.4%, and 81.8% of patients were estimated to be alive at 5, 10, and 15 years, respectively. Forty-nine patients died (9.5%), following an infection in 14 cases (2.7%), natural causes in 14 (2.7%), cardiovascular events in 13 (2.5%), a second neoplasm in 6 (1.2%), and progression of HCL in 2 cases (0.4%). Following treatment of HCL with 2CDA, 80% of patients are estimated to be alive 15 years after diagnosis.

Defining TCRγδ lymphoproliferative disorders by combined immunophenotypic and molecular evaluation.

Tγδ large granular lymphocyte leukemia (Tγδ LGLL) is a rare lymphoproliferative disease, scantily described in literature. A deep-analysis, in an initial cohort of 9 Tγδ LGLL compared to 23 healthy controls, shows that Tγδ LGLL dominant clonotypes are mainly public and exhibit different V-(D)-J γ/δ usage between patients with symptomatic and indolent Tγδ neoplasm. Moreover, some clonotypes share the same rearranged sequence. Data obtained in an enlarged cohort (n = 36) indicate the importance of a combined evaluation of immunophenotype and STAT mutational profile for the correct management of patients with Tγδ cell expansions. In fact, we observe an association between Vδ2/Vγ9 clonality and indolent course, while Vδ2/Vγ9 negativity correlates with symptomatic disease. Moreover, the 7 patients with STAT3 mutations have neutropenia and a CD56-/Vδ2- phenotype, and the 3 cases with STAT5B mutations display an asymptomatic clinical course and CD56/Vδ2 expression. All these data indicate that biological characterization is needed for Tγδ-cell neoplasm definition.

Identification of novel STAT5B mutations and characterization of TCRß signatures in CD4+ T-cell large granular lymphocyte leukemia.

CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) is a rare subtype of T-LGLL with unknown etiology. In this study, we molecularly characterized a cohort of patients (n = 35) by studying their T-cell receptor (TCR) repertoire and the presence of somatic STAT5B mutations. In addition to the previously described gain-of-function mutations (N642H, Y665F, Q706L, S715F), we discovered six novel STAT5B mutations (Q220H, E433K, T628S, P658R, P702A, and V712E). Multiple STAT5B mutations were present in 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, either coexisting in one clone or in distinct clones. Patients with STAT5B mutations had increased lymphocyte and LGL counts when compared to STAT5B wild-type patients. TCRß sequencing showed that, in addition to large LGL expansions, non-leukemic T cell repertoires were more clonal in CD4+ T-LGLL compared to healthy. Interestingly, 25% (15/59) of CD4+ T-LGLL clonotypes were found, albeit in much lower frequencies, in the non-leukemic CD4+ T cell repertoires of the CD4+ T-LGLL patients. Additionally, we further confirmed the previously reported clonal dominance of TRBV6-expressing clones in CD4+ T-LGLL. In conclusion, CD4+ T-LGLL patients have a typical TCR and mutation profile suggestive of aberrant antigen response underlying the disease.

How I treat refractory/relapsed hairy cell leukemia with BRAF inhibitors.

Hairy cell leukemia (HCL) responds very well to frontline chemotherapy with purine analogs (cladribine and pentostatine). However, approximately half of patients experience 1 or more relapses, which become progressively resistant to these myelotoxic and immunosuppressive agents. At progression, standard therapeutic options include a second course of purine analogs alone or in combination with rituximab and, upon second relapse, therapy with the anti-CD22 immunotoxin moxetumomab pasudotox. Furthermore, blockade of the mutant BRAF-V600E kinase (the pathogenetic hallmark of HCL) through orally available specific inhibitors (vemurafenib or dabrafenib) effaces the peculiar morphologic, phenotypic, and molecular identity of this disease and its typical antiapoptotic behavior and is emerging as an attractive chemotherapy-free strategy in various clinical scenarios. These include patients with, or at risk of, severe infections and, in a highly effective combination with rituximab, patients with relapsed or refractory HCL. Other treatments explored in clinical trials are BTK inhibition with ibrutinib and co-inhibition of BRAF (through dabrafenib or vemurafenib) and its downstream target MEK (through trametinib or cobimetinib). Here, we focus on our experience with BRAF inhibitors in clinical trials and as off-label use in routine practice by presenting 3 challenging clinical cases to illustrate their management in the context of all available treatment options.

Choroidal vasculature analysis in MEK inhibitor-associated retinopathy.

PURPOSE: To evaluate choroidal vascularity index (CVI) in patients developing mitogen-activated protein kinase kinase (MEK) inhibitor-associated retinopathy (MEKAR). METHODS: In this prospective observational study, extensive ophthalmic examination was performed, including enhanced-depth-imaging-optical coherence tomography (EDI-OCT). EDI-OCT scans of patients receiving Cobimetinib, taken at baseline and at MEKAR manifestation, were considered for choroid analysis. Choroidal thickness (CT) was measured on high-resolution b-scans passing through the fovea at three different locations. Same scans were therefore imported for binarization into a previously reported software and CVI was calculated as the ratio of luminal area (LA) to total choroid area (TCA). RESULTS: When compared to baseline, eyes with MEKAR (14 eyes) did not show significative CT variation in subfoveal region (p = 0,57), 750-µm-nasal to the fovea (p = 0,08) and 750-µm-temporal to the fovea (p = 0,07). Similarly, there were no statistically significant differences for TCA (p = 0.54), LA (p = 0.85), stromal area (SA) (p = 0.13), LA/SA (p = 0.34) and CVI (p = 0.47). Best-corrected visual acuity was significantly reduced at fluid accumulation when compared to baseline values (p = 0.03), with complete recovery after fluid resolution (p = 0.73). CONCLUSION: Multiple parameters reflecting the status of the choroid seemed not influenced by Cobimetinib administration. Retinal pigment epithelium toxic disfunction likely represents the crucial step in MEKAR pathogenesis.

A Curious Novel Combination of Nucleophosmin (NPM1) Gene Mutations Leading to Aberrant Cytoplasmic Dislocation of NPM1 in Acute Myeloid Leukemia (AML).

Nucleophosmin (NPM1) mutations occurring in acute myeloid leukemia (AML) (about 50 so far identified) cluster almost exclusively in exon 12 and lead to common changes at the NPM1 mutants C-terminus, i.e., loss of tryptophans 288 and 290 (or 290 alone) and creation of a new nuclear export signal (NES), at the bases of exportin-1(XPO1)-mediated aberrant cytoplasmic NPM1. Immunohistochemistry (IHC) detects cytoplasmic NPM1 and is predictive of the molecular alteration. Besides IHC and molecular sequencing, Western blotting (WB) with anti-NPM1 mutant specific antibodies is another approach to identify NPM1-mutated AML. Here, we show that among 382 AML cases with NPM1 exon 12 mutations, one was not recognized by WB, and describe the discovery of a novel combination of two mutations involving exon 12. This appeared as a conventional mutation A with the known TCTG nucleotides insertion/duplication accompanied by a second event (i.e., an 8-nucleotide deletion occurring 15 nucleotides downstream of the TCTG insertion), resulting in a new C-terminal protein sequence. Strikingly, the sequence included a functional NES ensuring cytoplasmic relocation of the new mutant supporting the role of cytoplasmic NPM1 as critical in AML leukemogenesis.