Predicting development of ipilimumab-induced hypophysitis: utility of T4 and TSH index but not TSH.

PURPOSE: Ipilimumab, a monoclonal antibody inhibiting CLTA-4, is an established treatment in metastatic melanoma, either alone or in combination with nivolumab, and results in immune mediated adverse events, including endocrinopathy. Hypophysitis is one of the most common endocrine abnormalities. An early recognition of hypophysitis may prevent life threatening consequences of hypopituitarism; therefore, biomarkers to predict which patients will develop hypophysitis would have clinical utility. Recent studies suggested that a decline in TSH may serve as an early marker of IH. This study was aimed at assessing the utility of thyroid function tests in predicting development of hypophysitis. METHODS: A retrospective cohort study was performed for all patients (n = 308) treated with ipilimumab either as a monotherapy or in combination with nivolumab for advanced melanoma at the Royal Marsden Hospital from 2010 to 2016. Thyroid function tests, other pituitary function tests and Pituitary MRIs were used to identify those with hypophysitis. RESULTS AND CONCLUSIONS: Ipilimumab-induced hypophysitis (IH) was diagnosed in 25 patients (8.15%). A decline in TSH was observed in hypophysitis cohort during the first three cycles but it did not reach statistical significance (P = 0.053). A significant fall in FT4 (P < 0.001), TSH index (P < 0.001) and standardised TSH index (P < 0.001) prior to cycles 3 and 4 in hypophysitis cohort was observed. TSH is not useful in predicting development of IH. FT4, TSH index and standardised TSH index may be valuable but a high index of clinical suspicion remains paramount in early detection of hypophysitis.

Neurotoxicity from immune-checkpoint inhibition in the treatment of melanoma: a single centre experience and review of the literature.

Background: Treatment with immune checkpoint inhibitors (ICPi) has greatly improved survival for patients with advanced melanoma in recent years. Anti-CTLA-4 and anti-PD1 antibodies have been approved following large Phase III trials. Immune-related neurological toxicity of varying severity has been reported in the literature. The cumulative incidence of neurotoxicity among ipilimumab, nivolumab and pembrolizumab is reported as <1% in published clinical trials. We aimed to identify the incidence of neurotoxicity in our institution across anti-CTLA4 and anti-PD-1 antibodies, including the combination of ipilimumab with nivolumab. We also review the existing literature and propose an investigation and management algorithm. Methods: All patients with advanced melanoma treated with ipilimumab, nivolumab, pembrolizumab or the combination of ipilimumab and nivolumab (ipi + nivo), managed at the Royal Marsden Hospital between September 2010 and December 2015, including patients on (published) clinical trials were included. Medical records for each patient were reviewed and information on neurotoxicity recorded. A systematic search strategy was performed to collate existing reports of neurological toxicity. Results: In total, 413 immunotherapy treatment episodes in 352 patients were included, with median follow-up of 26.7 months. Ten cases of neurotoxicity were recorded, affecting 2.8% of patients overall, ranging from grade 1 to 4, affecting both central and peripheral nervous systems. A rate of 14% was noted with ipi + nivo. Three of five patients commenced on corticosteroids responded to these. Six patients had made a full recovery at the time of reporting. A favorable radiological response was found in 7 of the 10 cases. Unusual presentations are described in detail. Conclusions: Neurological toxicity is not uncommon, and may be more frequent in patients treated with combination ipi + nivo. Patterns of presentation and response to treatment are varied. A prompt and considered approach is required to optimize outcomes in this group of patients.

Thyroid abnormalities following the use of cytotoxic T-lymphocyte antigen-4 and programmed death receptor protein-1 inhibitors in the treatment of melanoma.

CONTEXT: Checkpoint inhibitors are emerging as important cancer therapies but are associated with a high rate of immune side effects, including endocrinopathy. OBJECTIVE: To determine the burden of thyroid dysfunction in patients with melanoma treated with immune checkpoint inhibitors and describe the clinical course. DESIGN AND PATIENTS: Consecutive patients with melanoma treated with either ipilimumab, nivolumab, pembrolizumab or the combination of ipilimumab and nivolumab were identified. Baseline thyroid function tests were used to exclude those with pre-existing thyroid abnormalities, and thyroid function tests during treatment used to identify those with thyroid dysfunction. RESULTS: Rates of overt thyroid dysfunction were in keeping with the published phase 3 trials. Hypothyroidism occurred in 13·0% treated with a programmed death receptor-1 (PD-1) inhibitor and 22·2% with a combination of PD-1 inhibitor and ipilimumab. Transient subclinical hyperthyroidism was observed in 13·0% treated with a PD-1 inhibitor, 15·9% following a PD-1 inhibitor, and 22·2% following combination treatment with investigations suggesting a thyroiditic mechanism rather than Graves' disease, and a high frequency of subsequent hypothyroidism. Any thyroid abnormality occurred in 23·0% following ipilimumab, 39·1% following a PD-1 inhibitor and 50% following combination treatment. Abnormal thyroid function was more common in female patients. CONCLUSION: Thyroid dysfunction occurs commonly in patients with melanoma treated with immune checkpoint inhibitors, with rates, including subclinical dysfunction, occurring in up to 50%.

Serum lactate dehydrogenase as an early marker for outcome in patients treated with anti-PD-1 therapy in metastatic melanoma.

BACKGROUND: Treatment with programmed death receptor-1 (PD-1) antibodies is associated with high response rates in patients with advanced melanoma. Reliable markers for early response and outcome are still sparse. METHODS: We evaluated 66 consecutive patients with advanced/metastatic melanoma treated with nivolumab or pembrolizumab between 2013 and 2014. The main objectives of this study were to investigate whether, first, serum lactate dehydrogenase (LDH) at baseline (normal vs above the upper limit of normal) correlates with overall survival (OS), and, second, whether the change of LDH during treatment predicts response before the first scan and OS in patients with an elevated baseline LDH. RESULTS: After a median follow-up of 9 months, patients with an elevated baseline LDH (N=34) had a significantly shorter OS compared with patients with normal LDH (N=32; 6-month OS: 60.8% vs 81.6% and 12-month OS: 44.2% vs 71.5% (log-rank P=0.0292). In those 34 patients with elevated baseline LDH, the relative change during treatment was significantly associated with an objective response on the first scan: the 11 (32%) patients with partial remission had a mean reduction of -27.3% from elevated baseline LDH. In contrast, patients with progressive disease (N=15) had a mean increase of +39%. Patients with a relative increase over 10% from elevated baseline LDH had a significantly shorter OS compared with patients with ⩽ 10% change (4.3 vs 15.7 months, log-rank P<0.00623). CONCLUSIONS: LDH could be a useful marker at baseline and during treatment to predict early response or progression in patients with advanced melanoma who receive anti-PD-1 therapy.

Acute renal allograft rejection after immune checkpoint inhibitor therapy for metastatic melanoma.

Immune checkpoint inhibitors such as ipilimumab and nivolumab improve survival in patients with advanced melanoma and are increasingly available to clinicians for use in the clinic. Their safety in organ transplant recipients is not well defined but published case reports describing treatment with ipilimumab have not been complicated by graft rejection. No cases of anti-programmed cell death protein 1 administration are reported in this group. We describe a case of acute graft rejection in a kidney transplant recipient after treatment with nivolumab, after progression on ipilimumab. Potential factors increasing the risk of graft rejection in this case are discussed, in particular the contribution of nivolumab.

Updates in the pathogenesis and management of immune-related enterocolitis, hepatitis and cardiovascular toxicities.

Immune checkpoint inhibitors (ICIs) have become a cornerstone of treatment for many solid organ malignancies. Alongside increasing use, the occurrence of immune-related adverse events (irAEs) has also increased and remains a significant challenge when treating patients with ICI. The underlying pathophysiology of irAE development for many organ systems is yet to be elucidated, but may involve unmasking of latent autoimmunity, increased T-cell recognition of shared antigens on cancer and normal tissue and ICI-triggered immune dysregulation with overactivation of proinflammatory pathways and suppression of immune control pathways. Management strategies for irAEs have historically been borrowed from paradigms for conventional autoimmune conditions such as inflammatory bowel disease and autoimmune hepatitis; however, recent translational efforts have clearly demonstrated key differences in underlying immune signalling pathways. As we begin to understand these differences, we must adapt a more targeted approach to immunosuppression and exercise a more nuanced approach with the multiple biologic agents available to mitigate ICI-related toxicity without reversing the antitumour effect of ICI. In this review, we focus on three key immune-related toxicities where recent clinical and translational work has provided nuanced insights into pathogenesis and treatment strategies: enterocolitis, hepatitis and cardiovascular toxicity including myocarditis.

Developmental regulation of thymocyte susceptibility to deletion by "self"-peptide.

The specificity of the T cell receptor (TCR) repertoire for foreign peptide bound to self-major histocompatibility complex (MHC) molecules is determined in large part by positive and negative selection processes in the thymus, yet the mechanisms of these selection events remain unknown. Using in vitro organ culture of thymi isolated from mice transgenic for a TCR-alpha/beta specific for cytochrome c peptide bound to I-Ek, we analyzed the developmental timing of negative selection (deletion). On the basis of the experiments described below, we conclude that all CD4+8+ thymocytes, and only CD4+8+ thymocytes, are susceptible to negative selection mediated by the cytochrome c peptide antigen. First, we found that deletion of thymocytes resulting from addition of the cytochrome c peptide to the thymic organ cultures can occur at the earliest stage of TCR, CD4, and CD8 coexpression. Second, we found that CD4+8+ thymocytes isolated from positively selecting or nonselecting MHC haplotypes were equally efficiently deleted in vitro, suggesting that positive selection is not a prerequisite for deletion. Third, we examined the effects of TCR/ligand avidity on the developmental timing of deletion by varying the concentration of cytochrome c peptide added to the organ cultures. We detected deletion only at the CD4+8+ stage: intermediate concentrations of peptide that resulted in partial deletion of CD4+8+ cells did not eliminate the appearance of mature CD4+8- cells. Finally, we found that CD4+8- thymocytes were resistant to deletion as well as activation by peptide antigen added to the intact organ cultures. Nevertheless, the CD4+8- thymocytes isolated from the peptide-treated organ cultures responded vigorously to peptide presented by spleen cells in vitro. Thus, the T cells were tolerant of (but not anergized by) self-antigen encountered in thymic organ culture. Together, these results indicate that thymocytes susceptible to negative selection are not developmentally distinct from those susceptible to positive selection, and further, that the thymic microenvironment plays a role in regulating the outcome of TCR/ligand interactions.

Distinct roles of prostaglandin H synthases 1 and 2 in T-cell development.

Prostaglandin G and H synthases, or cyclooxygenases (COXs), catalyze the formation of prostaglandins (PGs). Whereas COX-1 is diffusely expressed in lymphoid cells in embryonic day 15.5 thymus, COX-2 expression is sparse, apparently limited to stromal cells. By contrast, COX-2 is predominant in a subset of medullary stromal cells in three- to five-week-old mice. The isozymes also differ in their contributions to lymphocyte development. Thus, experiments with selective COX-1 inhibitors in thymic lobes from normal and recombinase-activating gene-1 knockout mice support a role for this isoform in the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP). Concordant data were obtained in COX-1 knockouts. Pharmacological inhibition and genetic deletion of COX-2, by contrast, support its role during early thymocyte proliferation and differentiation and, later, during maturation of the CD4 helper T-cell lineage. PGE2, but not other PGs, can rescue the effects of inhibition of either isoform, although it acts through distinct EP receptor subtypes. COX-dependent PG generation may represent a mechanism of thymic stromal support for T-cell development.

Altered aging-related thymic involution in T cell receptor transgenic, MHC-deficient, and CD4-deficient mice.

During aging in mice and humans, a gradual decline in thymus integrity and function occurs (thymic involution). To determine whether T cell reactivity or development affects thymic involution, we compared the thymic phenotype in old (12 months) and young (2 months) mice transgenic for rearranged alphabeta or beta 2B4 T cell receptor (TCR) genes, mice made deficient for CD4 by gene targetting (CD4(-/-)), mice made deficient for major histocompatibility complex (MHC) class I (beta2M-/-) or class II genes (A(beta)(b-/-) on C57Bl/6 background) or both. The expected aging-related reductions in thymic weights were observed for all strains except those bearing disruption of both class I and class II MHC genes. Therefore, disruption of MHC class I and class II appeared to reverse or delay aging-related thymic atrophy at 12 months. Immunohistochemical analysis of aging-associated alterations in thymic morphology revealed that TCR alphabeta transgenes, CD4 disruption, and MHC class II disruption all reduced or eliminated these changes. All strains examined at 12 months showed alterations in the distribution of immature thymocyte populations relative to young controls. These results show that aging-associated thymic structural alterations, size reductions, and thymocyte developmental delays can be separated and are therefore causally unrelated. Furthermore, these results suggest that the T cell repertoire and/or its development play a role in aging-related thymic involution.

Illness perception and health-related quality of life in multiple sclerosis.

AIMS - A number of physical and psychological factors have been shown to affect health-related quality of life (HRQoL) in patients with multiple sclerosis (MS). Among these, the role of illness perceptions has not been established as an independent factor. This study, the first of its kind in an Australian population, aimed to use a large sample to determine the relative importance of individual factors to each domain of HRQoL, in particular the role of illness perception. MATERIALS AND METHODS - 580 patients with confirmed MS were assessed cross sectionally in a designated research clinic to determine the relative impact of physical factors (illness severity, duration, age, fatigue and pain) and psychological factors (mood, cognition and illness representations) on each domain of the SF-36. RESULTS - Categorical regression analysis showed that a combination of physical and psychological factors predicted 38-71% of variance in HRQoL. Illness perception was shown to have an independent effect on HRQoL in MS. The Extended Disability Status Scale was a significant determinant in all domains except for mental health. Depression was less prevalent than anxiety, but had a greater effect on function. CONCLUSION - Illness perception is an independent factor contributing to HRQoL in people with MS. Individual domains of HRQoL are associated with different patterns of physical and psychological factors. In the domains of role and social function, activities most highly valued by patients with MS, depression, anxiety, fatigue and illness perceptions are key determinants, all of which have the potential to be improved through specific interventions.

Quantitative analysis of the efficiency of clonal deletion in the thymus.

One of the major mechanisms for establishing self-tolerance is the clonal deletion of self-reactive T cells during their development in the thymus. Using a TCR transgenic mouse model, we have established a quantitative ex vivo assay for examining the sensitivity and specificity of negative selection. Thymic organ cultures established from mice of varying MHC haplotypes were incubated with antigen, and the efficiency of clonal deletion assessed. We show here that clonal deletion of CD4+8+ thymocytes is sensitive to both the gene dosage and the allelic variation of MHC class II molecules expressed on thymic antigen-presenting cells. We also find that when epithelial cells in the thymic cortex are the only antigen-presenting cells expressing the appropriate MHC class II molecules, negative selection of CD4+8+ cells is as efficient as when antigen is presented on all thymic antigen-presenting cells. These studies demonstrate that the induction of self-tolerance via clonal deletion in the thymus is a function not only of antigen concentration, but also of MHC class II cell-surface density. In addition, together with the reports of others, these results confirm that cortical epithelial cells can mediate negative selection, and demonstrate that they do so in the intact thymic microenvironment.

Purification and characterization of retrovirally transduced hematopoietic stem cells.

We have developed a method for the purification of retrovirally transduced hematopoietic stem cells, based on a modification of the stem cell purification protocols developed by Spangrude et al. [Spangrude, G., Heimfeld, S. & Weissman, I. (1988) Science 241, 58-64] and Spangrude and Scollay [Spangrude, G. & Scollay, R. (1990) Exp. Hematol. 18, 920-926], that depends upon the use of bone marrow cells isolated from 5-fluorouracil-treated mice that have been subsequently cocultivated with recombinant retrovirus-producing cell lines. We found that purified cell populations bearing a Sca 1+ Lin- Thy 1- surface phenotype represent a 50-100% pure population of spleen colony-forming cells (CFU-S day 12). Animals injected with 300 or more purified cells were consistently radioprotected and reconstituted in multiple lineages with donor cells. Sca 1+ Lin- Thy 1- CFU-S day 12 stem cells were shown to be efficiently (100%) transduced by the recombinant retroviruses used in the study. Gene transfer into long-term reconstituting stem cells, as evidenced by Southern blot analysis of mature hematopoietic cell types 3 months after transplantation, was observed only in recipients injected with large numbers (approximately 4000-5000) of the purified cells. The development of methods for purifying retrovirally transduced stem cells should prove extremely useful for various studies in which it is of interest to characterize the activity of a specific gene product (e.g., growth factor, receptor, oncogene) specifically in primitive hematopoietic cell types.

Do the CD4 and CD8 lineages represent parallel pathways?

It is now well established that the progression of T cell development requires interactions between the surfaces of thymocytes and thymic stromal cells. For example, the maturation of CD4+8+ cells into functional CD4+ or CD8+ cells requires TCR/MHC interactions, which, depending on the specificity of the particular TCR, direct the thymocyte to the appropriate lineage. Beyond this, little is known about the molecular mechanism of this lineage choice. Here we describe our recent studies of CD4/CD8 lineage commitment using TCR transgenic mice expressing a well-defined MHC class II specific TCR. While the results of these experiments are inconsistent with a model in which CD4 versus CD8 lineage is determined by an initial TCR/MHC/co-receptor interaction, they also do not support a simple stochastic model of lineage commitment. Instead, we suggest that the CD4 and CD8 lineage may not represent equivalent pathways of T cell maturation. Additionally, we draw several parallels between stochastic models in positive selection and in hematopoiesis.

PU.1 is required for myeloid-derived but not lymphoid-derived dendritic cells.

The ets-family transcription factor PU.1 is required for the proper development of both myeloid and lymphoid progenitors. We used PU. 1-deficient animals to examine the role of PU.1 during dendritic cell development. PU.1(-/-)animals produce lymphoid-derived dendritic cells (DC): low-density class II major histocompatibility complex [MHC-II(+)] CD11c(+) CD8alpha(+) DEC-205(+). But they lack myeloid-derived DC: low-density MHC-II(+) CD11c(+) CD8alpha(-) DEC-205(-). PU.1(-/-) embryos also lack progenitors capable of differentiating into myeloid DC in response to granulocyte-macrophage colony-stimulating factor plus interleukin-4. The appearance of lymphoid DC in developing PU.1(-/-)thymus was initially delayed, but this population recovered to wild type (WT) levels upon organ culture of isolated thymic lobes. PU. 1(-/-)lymphoid DC were functionally equivalent to WT DC for stimulating T-cell proliferation in mixed lymphocyte reactions. These results demonstrate that PU.1 is required for the development of myeloid DC but not lymphoid DC.

The COX-2 inhibitor NS-398 causes T-cell developmental disruptions independent of COX-2 enzyme inhibition.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the function of cyclooxygenases, COX-1 and COX-2, which catalyze the first step in the synthesis of inflammatory mediators (PGE2). We sought to understand the roles of cyclooxygenases and NSAIDs in T-cell development. Our data show no significant defects in T-cell development in fetal thymic organ cultures of mice disrupted in both or either COX genes or in mice disrupted in either EP-1 or EP-2 receptor genes. On the other hand, NSAIDs reproducibly caused thymocyte developmental defects. However, the specific effects of the COX-2 inhibitors were not correlated with their potency for inhibition of COX-2 activity. We focused on the NS-398 COX-2 inhibitor and showed that its effects could not be reversed by exogenous PGE2. Furthermore, NS-398 was inhibitory even when its target, COX-2, was absent. These data show that the T-cell developmental effects of NS-398 are COX-2 and PGE2 independent.

A peptide antigen antagonist prevents the differentiation of T cell receptor transgenic thymocytes.

The developmental fate of an immature T cell is determined in the thymus. Depending on the specificity of its TCR, a thymocyte receives signals to either die or differentiate. We have used fetal thymic organ cultures derived from TCR transgenic mice to examine the role of MHC/peptide ligands in T cell selection. Single amino acid substituted peptide analogues of the Ag recognized by the transgenic TCR were examined for their ability to enhance or interfere with positive selection. We have identified a nonstimulatory peptide analogue that interferes with the differentiation of transgenic CD4+8+ thymocytes into CD4+8- cells. We also show that this peptide, substituted in a TCR contact residue, is a competitive antagonist for activation of the T cell hybridoma expressing the same TCR. These observations demonstrate a novel mechanism for tolerance induction in the thymus.