Teresa Marafioti

Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

The cellular ancestry of tumor antigens One contributing factor in antitumor immunity is the repertoire of neoantigens created by genetic mutations within tumor cells. Like the corresponding mutations, these neoantigens show intratumoral heterogeneity. Some are present in all tumor cells (clonal), and others are present in only a fraction of cells (subclonal). In a study of lung cancer and melanoma, McGranahan et al. found that a high burden of clonal tumor neoantigens correlated with improved patient survival, an increased presence of tumor-infiltrating lymphocytes, and a durable response to immunotherapy. Science, this issue p. 1463 Analysis of the cellular ancestry of tumor neoantigens can predict which are most likely to induce an immune response. As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8+ tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non–small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy–induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens.

Ultra-deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas.

The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α‐ and β‐chains (TCRb). Our aim was to assess whether ultra‐deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples can provide insights into the clonality and heterogeneity of intratumoural T cells in ccRCCs, a tumour type that can display extensive genetic intratumour heterogeneity (ITH). For this purpose, DNA was extracted from two to four tumour regions from each of four primary ccRCCs and was analysed by ultra‐deep TCR sequencing. In parallel, tumour infiltration by CD4, CD8 and Foxp3 regulatory T cells was evaluated by immunohistochemistry and correlated with TCR‐sequencing data. A polyclonal T cell repertoire with 367–16 289 (median 2394) unique TCRb sequences was identified per tumour region. The frequencies of the 100 most abundant T cell clones/tumour were poorly correlated between most regions (Pearson correlation coefficient, –0.218 to 0.465). 3–93% of these T cell clones were not detectable across all regions. Thus, the clonal composition of T cell populations can be heterogeneous across different regions of the same ccRCC. T cell ITH was higher in tumours pretreated with an mTOR inhibitor, which could suggest that therapy can influence adaptive tumour immunity. These data show that ultra‐deep TCR‐sequencing technology can be applied directly to DNA extracted from unfractionated tumour samples, allowing novel insights into the clonality of T cell populations in cancers. These were polyclonal and displayed ITH in ccRCC. TCRb sequencing may shed light on mechanisms of cancer immunity and the efficacy of immunotherapy approaches. Copyright

Follicular peripheral T-cell lymphoma expands the spectrum of classical Hodgkin lymphoma mimics.

Epstein-Barr virus (EBV)-infected B cells with Reed-Sternberg–like cell (RS) features may occur in peripheral T-cell lymphomas (PTCLs), especially in angioimmunoblastic T-cell lymphoma. Here, we report 5 patients presenting with lymphadenopathy whose first biopsies demonstrated nodular lymphoid proliferations containing scattered CD30+, CD15+, EBV+ Hodgkin and Reed-Sternberg–like cells, which led to an initial diagnosis of lymphocyte-rich classical Hodgkin lymphoma. However, the uncommon clinical features and/or the occurrence of relapse as PTCL prompted review of the biopsies with expanded immunohistologic and molecular studies and revision of the diagnoses to follicular variant of PTCL (F-PTCL). All cases had atypical small to medium-sized CD3+ T cells that expressed CD10 (4/5) and the follicular helper T-cell (TFH) antigens BCL6, PD1, CXCL13, and ICOS. All demonstrated clonal T cells with a similar pattern in multiple samples from 4 patients. In 2 cases, flow cytometry demonstrated circulating lymphocytes with an abnormal sCD3−, CD4+, ICOS+ immunophenotype. Two patients had a skin rash at presentation, and 1 had B symptoms. Two of the 4 patients treated with polychemotherapy are alive at 3 and 6 years after first diagnosis. These cases highlight how some F-PTCLs may closely mimic lymphocyte-rich classical Hodgkin lymphoma requiring careful assessment of the T cells before rendering the latter diagnosis. The functional properties of TFH cells might lead to the presence of EBV-positive B blasts with RS-like features in TFH-derived PTCL such as angioimmunoblastic T-cell lymphoma and F-PTCL.

BRAF V600E mutation‐specific antibody, a sensitive diagnostic marker revealing minimal residual disease in hairy cell leukaemia

Mondor, Assistance Publique-Hôpitaux de Paris, Universit e Paris 11, Department of Pathology, Hôpital Saint-Louis, Assistance PubliqueHôpitaux de Paris, Universit e Paris 7-Diderot, Haematology/Oncology, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Universit e Paris 7-Diderot, Inserm U728, Bone marrow transplantation unit, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Universit e Paris 7-Diderot, Department of Pharmacology, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Universit e Paris 7-Diderot, and Department of Radiology, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Universit e Paris 7-Diderot, Paris, France E-mail: pauline.brice@sls.aphp.fr

Genome-wide analysis of pediatric-type follicular lymphoma reveals low genetic complexity and recurrent alterations of TNFRSF14 gene

Pediatric-type follicular lymphoma (PTFL) is a variant of follicular lymphoma (FL) with distinctive clinicopathological features. Patients are predominantly young males presenting with localized lymphadenopathy; the tumor shows high-grade cytology and lacks both BCL2 expression and t(14;18) translocation. The genetic alterations involved in the pathogenesis of PTFL are unknown. Therefore, 42 PTFL (40 males and 2 females; mean age, 16 years; range, 5-31) were genetically characterized. For comparison, 11 cases of conventional t(14:18)(-) FL in adults were investigated. Morphologically, PTFL cases had follicular growth pattern without diffuse areas and characteristic immunophenotype. All cases showed monoclonal immunoglobulin (IG) rearrangement. PTFL displays low genomic complexity when compared with t(14;18)(-) FL (mean, 0.77 vs 9 copy number alterations per case; P <001). Both groups presented 1p36 alterations including TNFRSF14, but copy-number neutral loss of heterozygosity (CNN-LOH) of this locus was more frequently observed in PTFL (40% vs 9%; P =075). TNFRSF14 was the most frequently affected gene in PTFL (21 mutations and 2 deletions), identified in 54% of cases, followed by KMT2D mutations in 16%. Other histone-modifying genes were rarely affected. In contrast, t(14;18)(-) FL displayed a mutational profile similar to t(14;18)(+) FL. In 8 PTFL cases (19%), no genetic alterations were identified beyond IG monoclonal rearrangement. The genetic landscape of PTFL suggests that TNFRSF14 mutations accompanied by CNN-LOH of the 1p36 locus in over 70% of mutated cases, as additional selection mechanism, might play a key role in the pathogenesis of this disease. The genetic profiles of PTFL and t(14;18)(-) FL in adults indicate that these are two different disorders.

Detection of LIM domain only 2 (LMO2) in normal human tissues and haematopoietic and non-haematopoietic tumours using a newly developed rabbit monoclonal antibody

Agostinelli C, Paterson J C, Gupta R, Righi S, Sandri F, Piccaluga P P, Bacci F, Sabattini E, Pileri S A & Marafioti T 
(2012) Histopathology 61, 33–46

Nodal reactive and neoplastic proliferation of monocytoid and marginal zone B cells: an immunoarchitectural and molecular study highlighting the relevance of IRTA1 and T‐bet as positive markers

Marginal zone B cells (MZCs) and monocytoid B cells (MBCs) appear to be related lymphoid cells that take part in reactive and neoplastic marginal zone proliferations. These lesions are not yet well characterized, and the aim of this study was to find better diagnostic criteria for them.

Another look at follicular lymphoma: Immunophenotypic and molecular analyses identify distinct follicular lymphoma subgroups

The aim of this study was to analyse the immunophenotypic and molecular features of a large series of follicular lymphomas, focusing in particular on atypical cases that fail to express CD10 and/or bcl‐2. Such cases present diagnostic pitfalls, especially with regard to the differential diagnosis from follicular hyperplasia and marginal zone B‐cell lymphoma. Therefore, we also included an immunohistochemical evaluation of stathmin, which is strongly expressed by germinal centre B cells, as a putative new marker for follicular lymphomas, particularly those with an atypical phenotype.

LLT1 and CD161 Expression in Human Germinal Centers Promotes B Cell Activation and CXCR4 Downregulation

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones, light and dark, with coordinated roles, controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells, but its functional role in the GC reaction has not been explored. In this study, we report high expression of LLT1 on GC-associated B cells, early plasmablasts, and GC-derived lymphomas. LLT1 expression was readily induced via BCR, CD40, and CpG stimulation on B cells. Unexpectedly, we found high expression of the LLT1 ligand, CD161, on follicular dendritic cells. Triggering of LLT1 supported B cell activation, CD83 upregulation, and CXCR4 downregulation. Overall, these data suggest that LLT1–CD161 interactions play a novel and important role in B cell maturation within the GC in humans.

ALDH, CA I, and CD2AP: novel, diagnostically useful immunohistochemical markers to identify erythroid precursors in bone marrow biopsy specimens.

Neoplastic erythroid proliferations may represent a diagnostic challenge owing to the difficulty in characterizing immature erythroblasts. Immunohistochemical expression of aldehyde dehydrogenase (ALDH), carbonic anhydrase isoenzyme I (CA I), and CD2-associated protein (CD2AP) was assessed in 66 bone marrow biopsy specimens and compared with glycophorin A and E-cadherin. ALDH, CA I, and CD2AP labeled neoplastic erythroblasts in most acute erythroid leukemias (AELs) and myelodysplasias and highlighted benign erythroid precursors within normal marrows, erythroid hyperplasias, acute lymphoblastic leukemias (ALLs), blastic plasmacytoid dendritic cell neoplasm, and most acute myeloid leukemias (AMLs). In 2 AELs, CD2AP was negative, and 1 AML lacked identifiable ALDH+ erythroid precursors. Immature erythroblasts were strongly ALDH+, weakly CA I+, weakly CD2AP±, E-cadherin±, and weakly glycophorin A±. AML was uncommonly weakly positive for ALDH, CA I, and CD2AP, and lymphoblasts from 1 ALL were weakly ALDH+. ALDH, CA I, and CD2AP are sensitive and relatively specific immunohistochemical markers for the erythroid lineage. ALDH is superior to glycophorin A and E-cadherin in highlighting immature erythroblasts.