Oncogenic alterations in KIR3DL1 in cutaneous acral CD8+ lymphoproliferative disorder.

BACKGROUND: Primary cutaneous acral CD8+ T-cell lymphoproliferative disorder (TLPD) is a rare and indolent lymphoma entity. Although known for years, the molecular pathogenesis is still unknown. OBJECTIVES: In order to better understand the molecular pathogenesis of cutaneous acral CD8+ TLPD and to identify further discriminatory markers to discern this lymphoma subtype from further CD8+ cutaneous lymphomas, we analysed five cases of cutaneous acral CD8+ TLPD for putative molecular alterations. METHODS: Somatic alterations were assessed by whole exome and targeted sequencing of paraffin-embedded tissue. Results were evaluated by immunohistochemical staining of respective relevant proteins. CD8+ cutaneous T-cell lymphomas (n = 12) served as control for KIR3DL1-staining. RESULTS: Copy number variations (CNV) analysis revealed a homozygous deletion of the KIR3DL1 gene in two of the analysed cases. This resulted in loss of KIR3DL1 protein expression which was observed in all cases of cutaneous acral CD8+ TLPD. In contrast, KIR3DL1 expression was more variable in other CD8+ cutaneous T-cell lymphomas with 50% of analysed cases (n = 12) being positive. In addition, one further case of acral CD8+ TLPD harboured a loss of function mutation in the PIK3R1 gene presumably activating the PI3K-AKT pathway. CONCLUSION: Alterations of KIR3DL1 gene may be of pathogenetic relevance for acral CD8+ TLPD. Loss of KIR3DL1 protein expression may support the diagnosis of this indolent lymphoma entity, albeit not being a subtype-specific discriminative feature.

Recurrent Alterations in the MAPK pathway in Sporadic Pyogenic Granuloma of Childhood.

Pyogenic granuloma is one of the most common vascular tumours. The cause of pyogenic granuloma was previously thought to be an inflammatory reaction with consecutive stimulation of endothelial cell proliferation. However, recent studies suggest that pyogenic granuloma may be driven by constitutive activation of the mitogen-activated protein kinase pathway. The aim of this study was to investigate the molecular profile of sporadic pyogenic granuloma of childhood, using a systematic approach scrutinizing potential aberrations within different oncogenic pathways. Within a retrospective setting pyogenic granuloma of 15 patients was analysed by targeted next generation sequencing using the Oncomine Focus Assay, which includes genes of key tumorigenic signalling pathways. Activating mutations were found in 4 out of 15 cases (27%). Two HRAS hotspot mutations (p.Gly13Arg, p.Ala59Thr), 1 BRAF (p.Val600Glu) mutation and a novel, previously not reported, MAP2K1 hotspot mutation (p.Glu203Lys) were identified. It is notable that all of these genes are involved in constitutive mitogen- activated protein kinase signalling. This study increases the range of underlying genetic alterations in pyogenic granuloma by identifying novel oncogenic mutations in crucial mitogen-activated protein kinase pathway genes. The results provide supporting evidence that activated mitogen-activated protein kinase signalling is a key driver in the pathogenesis of pyogenic granuloma, which might be exploited by targeted treatment approaches for selected cases.

EBER in situ hybridization in subcutaneous aluminum granulomas/lymphoid hyperplasia: A diagnostic clue to differentiate injection-associated lymphoid hyperplasia from other forms of pseudolymphomas and cutaneous lymphomas.

BACKGROUND: Subcutaneous vaccination or desensitization may induce persistent nodules at the injection sites. Without the knowledge of prior injection, histopathological work-up may be challenging. OBJECTIVE: Aim of this study was to contribute to the histopathological work-up of unclear subcutaneous nodules, especially their differentiation from cutaneous lymphoma. METHODS: We retrospectively reviewed clinical data and histopathological slides of four patients with subcutaneous nodules, which were suspected to suffer from cutaneous T- or B-cell lymphoma. Sections of these cases and 12 negative controls were stained with hematoxylin and eosin and a standardized immunohistochemical panel of B- and T-cell markers including EBER in situ hybridization as well as electron microscopy. RESULTS: In all cases, large histiocytes with granular cytoplasm compatible with intracellular aluminum hydroxide were present. EBER in situ hybridization revealed positive staining of these granular histiocytes while staining was absent in negative controls. LIMITATIONS: Post hoc completion of medical history revealed that vaccination or specific immunotherapy had been applied before at the biopsy site in only three out of four patients; one patient was lost to follow-up. CONCLUSION: EBER in situ hybridization is an adjunctive tool to differentiate aluminum-induced granuloma/lymphoid hyperplasia from other forms of pseudolymphoma and cutaneous B- or T-cell lymphomas.

SYK expression in monomorphic epitheliotropic intestinal T-cell lymphoma.

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), formerly known as type II enteropathy associated T-cell lymphoma (type II EATL), is a rare, aggressive primary intestinal T-cell lymphoma with a poor prognosis and an incompletely understood pathogenesis. We collected 40 cases of MEITL and 27 cases of EATL, formerly known as type I EATL, and comparatively investigated the T-cell receptor (TCR) itself and associated signaling molecules using immunohistochemistry, amplicon deep sequencing and bisulfite pyrosequencing. The TCR showed both an αß-T-cell origin (30%) and a γδ-T-cell derivation (55%) resulting in a predominant positive TCR phenotype in MEITL compared with the mainly silent TCR phenotype in EATL (65%). The immunohistochemical expression of the spleen tyrosine kinase (SYK) turned out to be a distinctive feature of MEITL (95%) compared with EATL (0%). Aberrant SYK overexpression in MEITL is likely caused by hypomethylation of the SYK promoter, while no common mutations in the SYK gene or in its promoter could be detected. Using amplicon deep sequencing, mutations in DNMT3A, IDH2, and TET2 were infrequent events in MEITL and EATL. Immunohistochemical expression of linker for activation of T-cells (LAT) subdivided MEITL into a LAT expressing subset (33%) and a LAT silent subset (67%) with a potentially earlier disease onset in LAT-positive MEITL.

Paraproteinaemia in Primary Cutaneous Marginal Zone Lymphoma.

Primary cutaneous marginal zone lymphomas (PCMZL) frequently exhibit lymphoplasmacytoid/plasmacytic differentiation, implying the capacity to produce monoclonal immunoglobulins. As these paraproteins are secreted, and thus are measurable in blood and urine, they may correlate with disease burden and serve as tumour markers reflecting therapeutic response. This study retrospectively analysed the records of 23 patients with PCMZL. During treatment and follow-up, laboratory tests, including full blood count, lactate dehydrogenase, serum protein electrophoresis and turbidimetric analyses, were conducted. Thirty-nine percent of cases showed a suspicious serum protein electrophoresis in terms of paraproteinaemia. In 44% of cases the heavy and light chain restriction in tissue samples correlated with serological findings. Altogether, 89% of the PCMZL patients with paraproteinaemia eventually experienced a relapse, in contrast to 62% of the group without paraproteinaemia. This study analysed the incidence and clinical implications of paraproteinaemia in patients with PCMZL. A clear correlation was found between paraproteinaemia, tumour relapse and therapeutic intervention.

Alternative BRAF mutations in BRAF V600E-negative hairy cell leukaemias.

The BRAF V600E mutation in exon 15 is considered the disease-defining mutation in hairy cell leukaemia (HCL), but single HCL cases lacking this mutation have been described. In 24 HCL, as well as in 194 various mature B- and T-cell neoplasms, we extended the search for BRAF mutations to exon 11. Two V600E-negative HCL contained novel, potentially functionally relevant mutations in exon 11 (F468C and D449E), while one other HCL was BRAF wild-type in exons 2-17. All non-HCL lymphomas lacked BRAF mutations. We therefore suggest screening of BRAF V600E-negative HCL for alternative exon 11 mutations in the diagnostic setting.

The immune response to a Coxiella burnetii vaccine in sheep varies according to their natural pre-exposure.

Q fever in humans is caused by Coxiella (C.) burnetii. In 2008 and 2012, cases of Q fever in humans were linked to an infected flock of approximately 650 ewes. Since 2013 gimmers (G'13, G'14, G'15 etc.) were primary vaccinated (two doses) with an inactivated C.burnetii vaccine without any revaccination. In 2013, 30 ewes were primary vaccinated (A'13). Shedding was annually monitored by qPCR-testing of vaginal and nasal swabs collected at lambing. Animals were tested for Phase I- (PhI) and PhII-antibodies (Ab) and for PhII-specific-interferon-γ (IFN-γ) before and after vaccination. The effect of a revaccination was determined in 2018 and 2023. Groups of randomly selected gimmers primary vaccinated in 2015, 2016 and 2017 and a mixed group of older animals (A'13, G'13 and G'14) were revaccinated once in 2018. The trial was repeated in 2023 on groups primary vaccinated in 2019-2023. Major shedding after the outbreak in 2012 ceased in 2014. Thereafter C.burnetii was only sporadically detected at low-level in 2018, 2021 and 2023. Sheep naturally exposed to C.burnetii during the outbreak in 2012 (A'13, G'13) mounted a strong and complete (PhI, PhII, IFN-γ) recall immune response after vaccination. A serological PhI+/PhII+ pattern dominated after vaccination. In contrast, since 2014 a weaker immune response (PhII-titre, IFN-γ) and a dominance of the PhI-/PhII+ pattern was observed in vaccinated gimmers. The number of serologically non-responding gimmers to vaccination increased to 25.0 % in G'16/G'17 and 40.4 % in G'19/G'20. But revaccination even three (G'15 in 2018) and four (G'19 in 2023) years after primary vaccination resulted in a strong and complete immune response. No difference of the immune response nor to more recently primary vaccinated animals (G'23 in 2023) nor to those animals that were present during the outbreak (A'13/G'13/G'14 in 2018) was observed.

Primary cutaneous marginal zone lymphomas with plasmacytic differentiation show frequent IgG4 expression.

The expression of IgG4 in malignant B-cell lymphomas has only partially been studied. Recent reports described single cases of marginal zone lymphomas arising in the ocular adnexae that express IgG4. Moreover, a subset of dura-associated marginal zone lymphomas appear to express IgG4 as well. We investigated IgG4 expression in a more systematic manner in a large cohort of marginal zone lymphoma specimens derived from the archive of our institute. Overall, we examined 169 marginal zone lymphomas of various primary sites that displayed a distinct plasmacytic differentiation and light chain restriction, allowing for a detailed investigation of the immunoglobulin heavy chain expression in these tumors by immunohistochemistry. Unexpectedly, primary cutaneous marginal zone lymphomas showed frequent IgG4 expression. Although only 1 out of 120 noncutaneous marginal zone lymphomas, located in the ocular adnexae, expressed IgG4, 19 of 49 (39%) primary cutaneous marginal zone lymphomas showed this feature, constituting the highest expression rate of IgG4 reported to date in any B-cell lymphoma. None of the IgG4-positive cutaneous marginal zone lymphomas with available clinical data showed evidence of a preexisting systemic IgG4-related disease, suggesting a localized immunologic IgG4-driven pathogenetic process at early stages of the disease. IgG4-positive and IgG4-negative primary cutaneous marginal zone lymphomas did not significantly differ in architectural features of the infiltrate or the composition of the reactive T-cell infiltrate as determined by analysis of T-cell content, CD4/CD8 ratio, and content of FOXP3- and PD1-positive T cells. Although the pathogenetic role of IgG4 expression in a significant subset of primary cutaneous marginal zone lymphomas with plasmacytic differentiation remains unclear at present, the demonstration of IgG4 expression in a marginal zone lymphoma involving the skin might be a helpful clue in the routine diagnostic setting, as these tumors will almost invariably be of primary cutaneous origin with an extremely low risk of spread to noncutaneous sites and an excellent prognosis.

Periocular cutaneous oncocytoma with signs of disrupted oxygen metabolism.

Oncocytomas are benign tumors most often occurring in salivary or lacrimal glands and thyroid tissue. As cutaneous oncocytoma is exceptionally rare, this tumor is uncommonly encountered by dermatopathologists. Herein, we illustrate the case of an 80-year-old man who presented with a slowly growing papule of the lower eyelid. Histopathologically, the adenomatous tumor was composed of large monomorphic cells with eosinophilic granular cytoplasm. Electron microscopy revealed abundant, enlarged and abnormally shaped mitochondria. These findings were consistent with an oncocytoma of the skin. The presented case is unique in that the thorough work-up of the tumor tissue revealed not only hyperplastic mitochondria, representing the ultrastructural correlate of the observed granular cytoplasm, but additionally disclosed functional consequences with elevated levels of reactive oxygen specimen (ROS) within the tumor. Disrupted oxygen metabolism may result from cellular aging processes and may putatively represent the underlying pathogenesis of oncocytoma.

Characterization of primary Wilms tumor cultures as an in vitro model.

Functional analysis of gene candidates and testing of novel therapeutics in Wilms tumors (WT) has been hampered by the lack of in vitro model systems. WT are characterized by a spectrum of histological appearances, but published cell lines are mostly derived from rare anaplastic variants or even non-WT. There has been some success in establishing primary cultures, but these are often poorly characterized or only derived from less frequent WT1 mutant tumors. We report the generation of a set of primary WT-cell cultures using a simple cultivation protocol. Our cultures could be established after preoperative chemotherapy and irrespective of histological subtypes or genetic alterations. The presence of tumor-specific genetic alterations validates these cultures as being tumor-derived. Genetic characterization is of utmost importance as some cultures with similar morphological appearance lacked such alterations and either represent clonal variants or normal cells. By immunohistochemistry, the cells are either epithelial or more mesenchymal, and the latter exhibiting a longer life span with 30 or more passages before undergoing senescence. This may be related to WT being embryonal tumors with a strong differentiation potential that may prevail in vitro. Telomeres progressively shorten with cultivation, but their length does not predict lifespan. hTERT transfection may partly allow establishment of immortalized lines, because 2/7 cultures avoid senescence even in later passages. Importantly, these cells can be efficiently manipulated by transfection, making them a useful model system for in vitro testing.

CXCR4-Directed Imaging and Endoradiotherapy in Desmoplastic Small Round Cell Tumors.

Desmoplastic small round cell tumor (DSRCT) is a rare, radiosensitive, yet difficult-to-treat sarcoma subtype affecting predominantly male adolescents. Extensive intraperitoneal seeding is common and requires multimodal management. With no standard therapy established, the prognosis remains poor, and new treatment options are needed. We demonstrate the clinical potential of C-X-C motif chemokine receptor 4 (CXCR4)-directed imaging and endoradiotherapy in DSRCT. Methods: Eight male patients underwent dual-tracer imaging with [18F]FDG and CXCR4-directed [68Ga]pentixafor PET/CT. A visual comparison of both tracers, along with uptake quantification in active DSRCT lesions, was performed. [68Ga]pentixafor uptake was correlated with immunohistochemical CXCR4 expression on tumor cells. Four patients with end-stage progressive disease underwent CXCR4-based endoradiotherapy. We report the safety, response by RECIST 1.1, and survival after endoradiotherapy. Results: Uptake of [68Ga]pentixafor in tumor lesions was demonstrated in all patients with DSRCT, providing diagnostic power comparable to [18F]FDG PET. Corresponding CXCR4 expression was confirmed by immunohistochemistry in all DSRCT biopsies. Finally, 4 patients were treated with CXCR4-directed [90Y]endoradiotherapy, 3 in a myeloablative dose range with subsequent autologous stem cell transplantation. All 3 required transfusions, and febrile neutropenia occurred in 2 patients (resulting in 1 death). Notably, severe nonhematologic adverse events were absent. We observed signs of response in all 3 patients, translating into disease stabilization in 2 patients for 143 and 176 d, respectively. In the third patient, postmortem autopsy confirmed a partial pathologic response. Conclusion: We validated CXCR4 as a diagnostic biomarker and a promising target for endoradiotherapy in DSRCT, demonstrated its feasibility, and provided the first evidence of its clinical efficacy.

Twenty-four months of bacterial colonialization and infection rates in patients with transcutaneous osseointegrated prosthetic systems after lower limb amputation-A prospective analysis.

Background: Transcutaneous osseointegrated prosthesis systems (TOPS) are alternative rehabilitation methods to socket prosthetics, after limb amputation. TOPS compromise a two-step surgery: starting with the implantation of the stem which is then followed by the creation of the transcutaneous stoma through which the exoprosthesis can be connected. Immediately after surgery, this opening is permanently exposed to pathogens. This study aimed to investigate the dynamics of bacterial colonization of the stoma to analyze whether obligate bacterial colonization leads to a risk of periprosthetic infections after TOPS treatment. Methods: This prospective study analyzed data from 66 patients (aged 26-75 years) after TOPS treatment between 2017 and 2019. Microbiological swabs from the stoma were analyzed on the first postoperative day and 3, 6, 12, and 24 months after stoma creation. Infection rates, laboratory values (CRP, leukocyte count, hemoglobin), and body temperature were recorded at these points in time. Statistical analysis was performed using SPSS 28. Results: The results show the formation of a stable environment dominated by Gram-positive bacteria in the stoma of TOPS patients over 24 months. Staphylococcus aureus, Staphylococcus spp., and Streptococcus spp. were the most common species found. With regard to the cohort up to the 3 months follow-up, 7.9% (five patients) developed infections surrounding the TOPS procedure. In relation to the whole cohort with loss to follow-up of 80.3% at the 24 months follow-up the infection rates increased up to 38.3%. Conclusion: The soft tissue inside and around the transcutaneous stoma is colonialized by multiple taxa and changes over time. A stable Gram-positive dominated bacterial taxa could be a protective factor for ascending periprosthetic infections and could possibly explain the relatively low infection rate in this study as well as in literature.

Panel Sequencing of Primary Cutaneous B-Cell Lymphoma.

BACKGROUND: Primary cutaneous follicular B-cell lymphoma (PCFBCL) represents an indolent subtype of Non-Hodgkin's lymphomas, being clinically characterized by slowly growing tumors of the skin and common cutaneous relapses, while only exhibiting a low propensity for systemic dissemination or fatal outcome. Up to now, only few studies have investigated underlying molecular alterations of PCFBCL with respect to somatic mutations. OBJECTIVES: Our aim was to gain deeper insight into the pathogenesis of PCFBCL and to delineate discriminatory molecular features of this lymphoma subtype. METHODS: We performed hybridization-based panel sequencing of 40 lymphoma-associated genes of 10 cases of well-characterized PCFBCL. In addition, we included two further ambiguous cases of atypical B-cell-rich lymphoid infiltrate/B-cell lymphoma of the skin for which definite subtype attribution had not been possible by routine investigations. RESULTS: In 10 out of 12 analyzed cases, we identified genetic alterations within 15 of the selected 40 target genes. The most frequently detected alterations in PCFBCL affected the TNFRSF14, CREBBP, STAT6 and TP53 genes. Our analysis unrevealed novel mutations of the BCL2 gene in PCFBCL. All patients exhibited an indolent clinical course. Both the included arbitrary cases of atypical B-cell-rich cutaneous infiltrates showed somatic mutations within the FAS gene. As these mutations have previously been designated as subtype-specific recurrent alterations in primary cutaneous marginal zone lymphoma (PCMZL), we finally favored the diagnosis of PCMZL in these two cases based on these molecular findings. CONCLUSIONS: To conclude, our molecular data support that PCFBCL shows distinct somatic mutations which may aid to differentiate PCFBCL from pseudo-lymphoma as well as from other indolent and aggressive cutaneous B-cell lymphomas. While the detected genetic alterations of PCFBCL did not turn out to harbor any prognostic value in our cohort, our molecular data may add adjunctive discriminatory features for diagnostic purposes on a molecular level.

Autophagy Blockage Reduces the Incidence of Pancreatic Ductal Adenocarcinoma in the Context of Mutant Trp53.

Macroautophagy (hereafter referred to as autophagy) is a homeostatic process that preserves cellular integrity. In mice, autophagy regulates pancreatic ductal adenocarcinoma (PDAC) development in a manner dependent on the status of the tumor suppressor gene Trp53. Studies published so far have investigated the impact of autophagy blockage in tumors arising from Trp53-hemizygous or -homozygous tissue. In contrast, in human PDACs the tumor suppressor gene TP53 is mutated rather than allelically lost, and TP53 mutants retain pathobiological functions that differ from complete allelic loss. In order to better represent the patient situation, we have investigated PDAC development in a well-characterized genetically engineered mouse model (GEMM) of PDAC with mutant Trp53 (Trp53 R172H ) and deletion of the essential autophagy gene Atg7. Autophagy blockage reduced PDAC incidence but had no impact on survival time in the subset of animals that formed a tumor. In the absence of Atg7, non-tumor-bearing mice reached a similar age as animals with malignant disease. However, the architecture of autophagy-deficient, tumor-free pancreata was effaced, normal acinar tissue was largely replaced with low-grade pancreatic intraepithelial neoplasias (PanINs) and insulin expressing islet ß-cells were reduced. Our data add further complexity to the interplay between Atg7 inhibition and Trp53 status in tumorigenesis.

Long-term control of Coxiellosis in sheep by annual primary vaccination of gimmers.

Coxiella (C.) burnetii, a Gram-negative intracellular bacterium, causes Q fever in humans and Coxiellosis in animals. Ruminants are a primary source of human infection with C.burnetii. In 2013, vaccination was implemented in a sheep flock with 650 ewes associated with two outbreaks of Q fever in humans in 2008 and 2012. Only gimmers (yearlings) received two doses of a commercial C.burnetii phase I whole cell vaccine three weeks apart (primary vaccination) without any revaccination. Vaginal and nasal swabs collected shortly after lambing were tested by qPCR. Additionally, a group of non-vaccinated sentinels was serologically monitored for phase I (PhI), II (PhII) antibodies and for Interferon γ (IFN-γ) after stimulation of whole blood cells with PhII-antigen with and without an IL-10-neutralizing monoclonal antibody. In 2021, 679 sera collected in 2014-2021 were retested retrospectively with three commercial ELISA kits and one batch of an in-house PhI/PhII-ELISA. A low-level shedding of C.burnetii (<103 mean C.burnetii/swab) was observed until 2014. In 2021 C.burnetii was detected in two animals (<103.1C.burnetii/swab), but vaginal swabs collected at two subsequent lambing seasons remained negative. Seroconversion of sentinels was detected until 2017. However, the retrospective analysis of sentinels in 2021 revealed additional single seropositive animals from 2018 to 2021. IFN-γ reactivity was observed during the whole study period; it peaked in 2014 and in 2018 and decreased thereafter. The sporadic detection of C.burnetii and the immune responses of sentinels suggested that a subliminal infection persisted despite vaccination. Nevertheless, vaccination of gimmers prevented the development of a major outbreak, it controlled the infection and reduced the risk of human infection.

Acute systemic knockdown of Atg7 is lethal and causes pancreatic destruction in shRNA transgenic mice.

The notion that macroautophagy/autophagy is a potentially attractive therapeutic target for a variety of diseases, including cancer, largely stems from pre-clinical mouse studies. Most of these examine the effects of irreversible and organ confined autophagy deletion using site specific Cre-loxP recombination of the essential autophagy regulating genes Atg7 or Atg5. Model systems with the ability to impair autophagy systemically and reversibly at all disease stages would allow a more realistic approach to evaluate the consequences of authophagy inhibition as a therapeutic concept and its potential side effects. Here, we present shRNA transgenic mice that via doxycycline (DOX) regulable expression of a highly efficient miR30-E-based shRNA enabled knockdown of Atg7 simultaneously in the majority of organs, with the brain and spleen being noteable exceptions. Induced animals deteriorated rapidly and experienced profound destruction of the exocrine pancreas, severe hypoglycemia and depletion of hepatic glycogen storages. Cessation of DOX application restored apparent health, glucose homeostasis and pancreatic integrity. In a similar Atg5 knockdown model we neither observed loss of pancreatic integrity nor diminished survival after DOX treatment, but identified histological changes consistent with steatohepatitis and hepatic fibrosis in the recovery period after termination of DOX. Regulable Atg7-shRNA mice are valuable tools that will enable further studies on the role of autophagy impairment at various disease stages and thereby help to evaluate the consequences of acute autophagy inhibition as a therapeutic concept.Abbreviations: ACTB: actin, beta; AMY: amylase complex; ATG4B: autophagy related 4B, cysteine peptidase; ATG5: autophagy related 5; ATG7: autophagy related 7; Cag: CMV early enhancer/chicken ACTB promoter; Col1a1: collagen, type I, alpha 1; Cre: cre recombinase; DOX: doxycycline; GCG: glucagon; GFP: green fluorescent protein; INS: insulin; LC3: microtubule-associated protein 1 light chain 3; miR30-E: optimized microRNA backbone; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; PNLIP: pancreatic lipase; rtTA: reverse tetracycline transactivator protein; SQSTM1/p62: sequestome 1; TRE: tetracycline responsive element.