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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 © 2013 Pathological Society of Great Britain and Ireland.

The long-term effects of antenatal dexamethasone treatment on brain remodelling in 3-month-old male Sprague Dawley rats whose mothers had been treated with dexamethasone were investigated in the present study. Dorsal hippocampus, basolateral amygdala and nucleus accumbens volume, cell numbers, and GFAP-immunoreactive astroglial cell morphology were analysed using stereology. Total brain volume as assessed by micro-CT was not affected by the treatment. The relative volume of the dorsal hippocampus (% of total brain volume) showed a moderate, by 8%, but significant reduction in dexamethasone-treated versus control animals. Dexamethasone had no effect on the total and GFAP-positive cell numbers in the hippocampal subregions, basolateral amygdala, and nucleus accumbens. Morphological analysis indicated that numbers of astroglial primary processes were not affected in any of the hippocampal subregions analysed but significant reductions in the total primary process length were observed in CA1 by 32%, CA3 by 50%, and DG by 25%. Mean primary process length values were also significantly decreased in CA1 by 25%, CA3 by 45%, and DG by 25%. No significant astroglial morphological changes were found in basolateral amygdala and nucleus accumbens. We propose that the dexamethasone-dependent impoverishment of hippocampal astroglial morphology is the case of maladaptive glial plasticity induced prenatally.

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Aims: 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. Methods and results: Two hundred and five follicular lymphomas were investigated with immunohistochemistry and fluorescence in-situ hybridization (FISH). The use of three distinct anti-bcl-2 antibodies together with CD10 expression data and FISH analysis for bcl-2 and bcl-6 rearrangements allowed subclassification of follicular lymphoma into four distinct subgroups: (i) CD10-positive/bcl-2-positive, (ii) CD10-positive/bcl-2-negative, (iii) CD10-negative/bcl-2-positive, and (iv) CD10-negative/bcl-2-negative. All cases were bcl-6-positive. STMN1 (stathmin) was shown to be helpful in diagnosing bcl-2-negative and/or CD10-negative follicular lymphomas, and in their distinction from marginal zone B-cell lymphoma. Conclusions: Combined immunohistological and molecular analyses reveal that follicular lymphomas showing an atypical immunophenotypic and molecular profile exist, and we demonstrate that STMN1 represents a novel useful diagnostic marker for these. © 2013 Blackwell Publishing Ltd.

ADCT-301, currently in Phase I clinical trial, is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human IL-2R-α (CD25) conjugated through a cleavable linker to a PBD dimer warhead with a drug-antibody ratio of 2.3. In vitro and ex vivo, ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and targeted cytotoxicity against a panel of human lymphoma cell lines. On release, PBD dimers bind in the DNA minor groove and exert their cytotoxic action via the formation of DNA interstrand cross-links. In vivo, ADCT-301 demonstrates dose-dependent antitumor activity against subcutaneous and disseminated lymphoma models. For example, in the Karpas 299 xenograft model, 10/10 tumor-free survivors are observed following a single dose of 0.5 mg/kg, whereas Adcetris® gives only a modest delay in mean tumor growth at 0.5 mg/kg, despite this tumor expressing three-fold higher target antigen levels for this drug. The current study aimed to further define the mechanism of action of ADCT-301 and validate pharmacodynamic assays for clinical development. In Karpas 299 cells, evidence for internalization of ADCT-301 was shown by a reduction of CD25 molecules on the cell surface over the first three hours post-treatment followed by a return to pre-treatment levels by 16 hours. This is consistent with the documented rapid recycling of CD25 to the membrane after exposure to IL-2 (Hemar et al Journal of Cell Biology 1995). Furthermore, ADCT-301 on the cell surface declined by >70% over four hours. Following a two-hour exposure to ADCT-301, DNA interstrand cross-linking, measured using a modification of the single cell gel electrophoresis (comet) assay, reached a peak between 4 and 8 hours after which cross-links persisted up to 36 hours. In contrast, the peak of cross-link formation for an equimolar concentration of warhead was immediately following drug exposure and a non-targeted PBD-containing ADC did not produce crosslinks in these cells. A strong correlation (r = 0.97) between loss of viability and DNA cross-link formation provides support for this DNA damage being the critical initiating mechanism of cytotoxicity of ADCT-301. We have previously shown that PBD-induced DNA interstrand cross-links elicit a robust, but delayed γ-H2AX response (Wu et al Clinical Cancer Research 2013). In Karpas 299 cells phosphorylation of H2AX was observed 24 hours after a two-hour exposure to sub-GI50 concentrations of ADCT-301. In these cells continuous exposure to ADCT-301 resulted in a dose-dependent G2/M arrest, peaking at 48 hours, later than for the naked warhead. The peak of the early apoptosis marker annexin-V on the cell surface of Karpas 299 cells was observed between 60 and 72 hours and maximal loss of viability was at 96 hours. Significant bystander killing of CD25-negative human Burkitt's lymphoma-derived Ramos cells was demonstrated for ADCT-301 both by co-culture experiments with CD25-positive Karpas 299 cells, and by media transfer from Karpas 299 cells treated with ADCT-301. This is important as many lymphomas are heterogeneous in their CD25 expression profile (Strauchen et al American Journal of Pathology 1987). In SCID mice with Karpas 299 subcutaneous tumors a single dose of ADCT-301 was administered at 0.2 or 0.6 mg/kg. 24 hours after treatment, excised tumors showed a dose proportional increase in intensity of membrane and cytoplasmic staining by an anti-PBD payload antibody. Cross-linking was determined as 23% (0.2 mg/kg) vs 49% (0.6 mg/kg) (p ≤ 0.01) reduction in Tail Moment using the comet assay and dose-dependent γ-H2AX formation measured by immunohistochemistry was observed. No cross-linking was observed in matched lymphocyte samples. These data confirm the mechanism of cell killing of ADCT-301 and provide relevant pharmacodynamic assays for use in the clinical development of PBD-based ADCs. Disclosures Flynn: Spirogen/Medimmune: Employment. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics: Employment. Tyrer:Spirogen/Medimmune: Employment. Williams:Spirogen/Medimmune: Employment. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Despite the immunologic functions of T-cell receptor signaling molecules being extensively investigated, their potential as immunohistochemical markers has been poorly explored. With this background, we evaluated the expression of 5 intracellular proteins-GADS, DOK2, SKAP55, ITK, and PKCα-involved in T-cell receptor signaling in normal and neoplastic hematologic tissue samples, using antibodies raised against fixation-resistant epitopes of the 5 molecules. All 5 antibodies were associated with normal T-cell differentiation. GADS, DOK2, SKAP55, and ITK turned out to be T-cell lineage-specific markers in the setting of lymphoid and myeloid precursor neoplasms but showed differential expression in peripheral T-cell lymphoma (PTCL) subtypes, being detected in PTCL/not otherwise specified (NOS) and angioimmunoblastic T-cell lymphoma but negative in anaplastic large cell lymphoma (ALCL). Peripheral B-cell lymphomas were consistently negative for ITK, with occasional cases showing expression of DOK2 and SKAP55, and a proportion (47%) of hairy cell leukemias were GADS. Notably, PKCα highlighted a defective antigen in both PTCL/NOS (6%) and angioimmunoblastic T-cell lymphoma (10%), mostly negative in ALCL, and was aberrantly expressed in classical Hodgkin lymphoma (65%), Burkitt lymphoma (48%), and plasma cell myeloma (48%). In conclusion, all five molecules evaluated play a role in T-cell differentiation in normal and neoplastic tissues. They can be applied confidently to routine sections contributing primarily to assignment of T-lineage differentiation in the setting of hematopoietic precursor neoplasms (GADS/DOK2/SKAP55/ITK) and for the differential diagnosis between ALCL and PTCL/NOS (GADS/DOK2/SKAP55/ITK) or classical Hodgkin lymphoma (PKCα). Finally, association with specific tumor subtypes may have therapeutic potential.