Aidan M. Rose

Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through BrafV600E or KrasG12D knockin) and TGFβ signalling ablation (through Tgfbr1 deletion) in LGR5+ve stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5+ve cells also results in cSCC development. These findings indicate that LGR5+ve stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis.

Thigh burns from exploding e-cigarette lithium ion batteries: First case series

E-cigarette (EC) use has risen meteorically over the last decade. The majority of these devices are powered by re-chargeable lithium ion batteries, which can represent a fire hazard if damaged, over-heated, over-charged or stored inappropriately. There are currently no reports in the medical literature of lithium ion battery burns related to EC use and no guidance on the appropriate management of lithium ion battery associated injuries. We report two individual cases of burn resulting from explosion of EC re-chargeable lithium ion batteries. Both patients required in-patient surgical management. We provide evidence that lithium ion battery explosions can be associated with mixed thermal and alkali chemical burns, resulting from the significant discharge of thermal energy and the dispersal of corrosive lithium ion compounds. We would recommend, as with other elemental metal exposures, caution in exposing lithium ion battery burns to water irrigation. Early and thorough cleaning and debridement of such burns, to remove residual lithium contamination, may limit the risk of burn wound extension and potentially improve outcomes.

E-cigarettes—beware of the rocket in your pocket

The recent Royal College of Physicians report on e-cigarettes and tobacco harm reduction missed an important point.1 2 We recently published the first case series of burn injuries from exploding rechargeable lithium ion batteries in e-cigarettes—thigh burns that required hospital admission and surgical debridement.3 Both patients described …

Adherence to burn center referral criteria--the major tip of the "minor" iceberg?

We read with interest the article by Carter et al, who revealed that 70% of their state’s acute burn admissions met American Burn Association referral criteria, yet 33% of these patients had no treatment in a specialist burn center. We presented similar work on a smaller scale, looking at adherence to British Burn Association referral criteria, but in a pediatric emergency department (ED). Our retrospective study included all patients presenting to the ED with a triage diagnosis of “Burn/ Scald” over 6 months. Of 190 patients, 126 or 66% (compared with 70% in Carter et al) presented with injuries meeting referral criteria. However, in our cohort, the number of these patients not referred to our burns service was significantly more, n 93 or 74% (33% in Carter et al). We then followed up this “under-referred” group of 93 patients to assess whether nonreferral results in harm. Eighty-four percent were reviewed and discharged in our ED and received no specialist input. Only three (3.2%) subsequently required referral to our burns centre; all were contact burns in children aged 5 years and were discharged without further intervention. Our studies are different in terms of not only patient groups but also the effect our respective health systems have on referral patterns. We have shown that there are many patients with perceived “minor” burns who slip through our EDs without referral. It is equally important to identify these patients with perceived “minor” burns who could benefit from early appropriate referral, given the documented risk of associated morbidities long-term. Strict adherence may result in a significant increase in workload for burn services with an as-yet-demonstrated benefit for these patients short-term. In our health service, for now, such criteria need to be interpreted with a degree of flexibility based on clinical judgment and experience, in coordination with burn centers, to help distribute such workload.

Reduced SMAD2/3 activation independently predicts increased depth of human cutaneous squamous cell carcinoma

The incidence of cutaneous squamous cell carcinoma (cSCC) is rising. Whilst the majority are cured surgically, aggressive metastatic cSCC carry a poor prognosis. Inactivating mutations in transforming growth factor beta (TGF-β) receptors have been identified amongst genetic drivers of sporadic tumours and murine models of cSCC, suggesting a tumour suppressor function for TGF-β in normal skin. However, paradoxically, TGF-β acts as a tumour promoter in some murine model systems. Few studies have analysed the role of TGF-β/activin signalling in human normal skin, hyper-proliferative skin disorders and cSCC. Antibodies recognising phospho-SMAD proteins which are activated during canonical TGF-β/activin signalling were validated for use in immunohistochemistry. A tissue microarray comprising FFPE lesional and perilesional tissue from human primary invasive cSCC (n=238), cSCC in-situ (n=2) and keratocanthoma (n=9) were analysed in comparison with tissues from normal human scalp (n=10). Phosphorylated SMAD2 and SMAD3 were detected in normal interfollicular epidermal keratinocytes and were also highly localised to inner root sheath, matrix cells and Keratin 15 positive cells. Lesional cSCC tissue had significantly reduced activated SMAD2/3 compared to perilesional tissue, consistent with a tumour suppressor role for SMAD2/3 activators in cSCC. Increased cSCC tumour thickness inversely correlated with the presence of phospho-SMADs in tumour tissue suggesting that a reduction in canonical TGF-β/activin signalling may be associated with disease progression.

Loss of TGF-β signaling drives cSCC from skin stem cells – More evidence

Tangible therapeutic advances for aggressive and potentially lethal forms of cutaneous squamous cell carcinoma (cSCC), one of the most common forms of cancer, are currently lacking. As our aging population continues to suffer from its relentless increase in incidence, defining the molecular events that drive cSCC, and importantly the context in which those events occur, clearly represent an urgent research need. Our recent data provides compelling support for the proposed tumor suppressor role of TGF-b signaling in skin tumourigenesis, as well as contextual insight into how loss of TGF-b signaling, when targeted to specific skin stem cell compartments, permits rapid tumor formation. In essence, evidence of a key tumor suppressor role for TGFb signaling in human squamo-proliferative disease and invasive cSCC already exists. Convincing reports include the genotypephenotype correlation between inactivating TGFBR1 mutation and familial multiple self-healing squamous epithelioma (MSSE), as well as reports of spontaneous cSCC arising secondary to systemic treatment with the pan-TGF-b ligand antibody (GC1008). In addition, TGF-b receptor mutations have also been detected in RAF-kinase induced skin tumors; which is where our study began. We initially performed targeted deep sequencing of 39 squamo-proliferative lesions from patients treated with vemurafenib (a BRAF inhibitor used to treat advanced melanoma) and identified mutations of both TGF-b receptors (TGFBR1 and TGFBR2) in 28% of lesions. Next, using the same targeted sequencing profile, we interrogated 91 sporadic human cSCC and 21 human primary cSCC cell lines and detected mutations of both TGF-b receptors in 43% of samples. Crucially, normal blood samples as well as matched normal distant and perilesional skin controls harboured no TGF-b receptor mutations, indicating that these mutations appeared to be lesion-specific non-germline events. Consolidating these findings, we detected a similar high frequency of TGF-b receptor mutations in a separate cohort of 30 cSCC analyzed by whole exome sequencing (WES) – with alterations in a total of 53% of samples analyzed using this platform with no mutations in matched normal samples. Factors such as varying efficiencies in deep sequencing techniques, an exceptionally high mutational burden of cSCC tumors (producing significant background passenger mutation rates) and tumor heterogeneity all contribute to the challenge of confirming driver gene status in cSCC. A robust analytical approach combining computational and statistical models, a functional in-vitro analysis of detected mutations and goldstandard in-vivo mouse models, not only provided substantial evidence to suggest these mutations were indeed driver mutations, but also the novel finding that Tgfbr1 deletion appears to drive tumourigenesis when specifically targeted to the bulge stem cell compartment of the hair follicle. A stringent combination of analytical software programs was used to predict the functional consequence of mutation. This approach predicted that 50% of TGFBR1 and 70% of detected TGFBR2 mutations were likely to be functionally damaging. The functional consequence of mutations detected by WES was further assessed by MutsigCV and IntOgen algorithms. Although these algorithms failed to detect TGFBR1 or TGFBR2 mutations as significant on an individual basis, it was apparent that this approach failed to account for the potential bias of detecting mutually exclusive mutations that may disrupt signaling pathways. IntOgen analysis also predicts the significance of mutation in signaling pathways and confirmed that the TGF-b signaling pathway was significantly mutated. In addition, analysis of variant allelic frequencies (VAF) demonstrated a significant proportion of tumors containing TGF-b receptor mutations exhibited the highest VAF’s in those mutations compare with VAF’s of other common drivers of disease, suggesting they were more likely to be initiating events. In strong supporting this hypothesis, clonal analysis of the WES data using ABSOLUTE predicted that 7 of the 8 TGF-b receptor mutations were indeed clonal and therefore likely to represent driver events. Functional evidence out-strips prediction programmes and algorithms. In-vitro TGF-b reporter gene analysis of a panel of TGF-b receptor mutants demonstrated a significant proportion of TGFBR1 mutants and all TGFBR2 mutants tested failed to restore active TGF-b signaling. In addition, restoring wild-type TGFBR2 receptor to TGFBR2null cSCC cells restored growth arrest. Taken together, these findings provide convincing evidence that loss of TGF-b tumor suppressor function is a common event in cSCC.

Abstract 1506: Frequent loss of function mutations in TGFβR1 and TGFβR2 identify hair follicle bulge stem cells as the cell of origin for cutaneous squamous cell carcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Solid tumors are typically considered to arise from the accumulation of mutations within either stem or differentiated cells and to evolve over several years. However, melanoma patients treated with inhibitors of oncogenic BRAF frequently present with keratoacanthomas and/or cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, possibly driven by paradoxical RAS/RAF MAPK pathway activation. Here, we identify frequent HRAS, TGFβR1 and TGFβR2 mutations in skin lesions from vemurafenib treated patients. Analysis of 98 human sporadic cSCC tumor samples and 21 cSCC cell lines revealed mutation of TGFβ receptors in 42% of samples and activating RAS mutations in 9% of samples. Functional analysis indicates that TGFβ receptor mutations frequently result in loss of canonical signaling. Analysis of normal human skin revealed localised TGFβ signaling in hair follicle bulge stem cells. In murine skin autocrine TGFβ signaling was highly localised to Lgr5+ve stem cells. We modelled hyperactivation of the MAPK pathway (through knockin of BRafV600E or KRASG12D) and the consequences of TGFβ signalling ablation (through the deletion of Tgfβr1) in Lgr5+ve cells. Whist BRaf or KRAS activation alone did not lead to cancer, homozygous deletion of Tgfβr1 enabled rapid cSCC development. Taken together, our results indicate that Lgr5+ve stem cells can act as the cell of origin for cSCC and that hyperactivation of the RAS-RAF-MAPK pathway, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis. Citation Format: Patrizia Cammareri, Aidan M. Rose, David F. Vincent, Silvana Libertini, Rachel A. Ridgway, Dimitris Athineos, Philip Coates, Angela McHugh, Celine Pourreyron, Jonas Larsson, Lindsay C. Spender, Gopal Sapkota, Karin Purdie, Charlotte Proby, Catherine A. Harwood, Irene M. Leigh, Hans Clevers, Nicholas Barker, Stefan Karlsson, Catrin Pritchard, Richard Marais, Andrew P. South, Owen J. Sansom, Gareth J. Inman. Frequent loss of function mutations in TGFβR1 and TGFβR2 identify hair follicle bulge stem cells as the cell of origin for cutaneous squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1506. doi:10.1158/1538-7445.AM2015-1506

Hemihypertrophy of a lower limb

elevated levels of mevalonic acid excreted in urine. Increased levels of IgD and, in most patients of IgA, in combination with enhanced excretion of mevalonic acid during febrile attacks provide strong suspicion for HIDS. The diagnosis is confirmed by demonstration of deficient MVK enzyme activity or by demonstration of disease-causing mutations. We present herein the case of two siblings who developed a periodic fever with pruritic rash during childhood. A diagnosis of HIDS seemed unlikely because of repeatedly normal levels of IgD and IgA in these patients. Although high serum IgD concentrations constitute a unique hallmark of this syndrome, 22% of patients’ IgD values are normal. In particular, IgD may be normal in patients under 3 years of age. In addition, an elevated serum IgD is not a specific marker for HIDS. Moreover, the level of serum IgD in HIDS does not correlate with disease severity or frequency of attacks. This suggests that IgD should not be a requirement for diagnosis, especially during the infancy, but can assist in making the diagnosis. The alterations of the immunoglobulin pattern in HIDS are not exclusively limited to IgD. Elevated immunoglobulin G and immunoglobulin M values have been found, and specially high IgA levels are found in the large majority of patients. There is no consensus for specific treatment for HIDS; some studies use drugs such as colchicine, steroids, simvastatin, anakinra, thalidomide and etanercept. In these cases, colchicine reduced the severity and frequency of both febrile attacks and episodes of generalised urticaria. Despite having taken a thorough family history and performed a careful clinical examination and despite the knowledge that normal levels of IgD do not exclude the presence of HIDS, the diagnosis of HIDS in this family might have been missed. A better awareness of periodic fevers among paediatricians is required at present, because HIDS syndrome, as other autoinflammatory syndromes and chronic diseases, adversely influences several aspects of quality of life In conclusion, HIDS is a challenging diagnosis to make, moreover when patients do not completely meet the criteria for the disease. Although genetic testing has been available since the end of the last millennium, there continued to be a median delay to diagnosis of 9.9 years after onset of symptoms. Remarkably, skin symptoms could be the first to appear, in the form of an urticarial rash, with exacerbations coinciding with episodes of fever, abdominal pain, enlarged lymph nodes and raised parameters of inflammation. An exhaustive evaluation of blood and urine biochemical data, a clinical examination during the attack and a close follow-up could suggest the correct diagnosis early and prevent unnecessary hospitalisations.