Dasatinib associated lymphadenopathy (DAL) is a rare adverse event in chronic myeloid leukemia patients (CML). A case of voluminous lymphadenopathy in the context of DAL is presented.A 40-year-old male patient was diagnosed with BCR-ABL1 positive chronic stage CML 2 years ago and achieved complete molecular response on nilotinib, which was switched to dasatinib due to nilotinib intolerance. After 5 months on dasatinib, the patient presented with a large mass in the axillary region.Common infectious and autoimmune etiologies of lymphadenopathy were ruled out. The positron emission tomography/computed tomography (PET/CT) demonstrated a hypermetabolic lymphadenopathy highly suspicious of lymphoma. The subsequent biopsy excluded lymphoma or extramedullary blastic transformation of CML and revealed reactive lymphadenopathy with mixed  pattern. Clinical history and clinicopathological correlation suggested the diagnosis of DAL.Dasatinib was discontinued and the patient remained in close follow-up. TKI treatment with nilotinib was reinitiated.Lymphadenopathy resolved clinically at 4 weeks and normalization of PET/CT findings was documented at 9 weeks after cessation of the drug. TKI treatment with nilotinib was reinitiated with good tolerance.DAL may present with voluminous lymphadenopathy consistent with malignancy in clinical and imaging workup. We describe the spectrum of lesions associated with DAL and identify common features with drug-induced lymphadenopathy. Some of these adverse events could be attributed to the limited specificity of the currently available TKIs resulting in the inhibition of other kinases, but further study is necessary to elucidate the underlying mechanisms.The safety profile varies among different agents and frequently the clinician must balance between an effective dose of a particular agent and the associated adverse events. Common adverse events include nausea/vomiting, diarrhea, muscle cramps, edema and cytopenias; less commonly severe vascular, cardiac, pulmonary, metabolic/endocrine and hepatic toxicities may arise. Herein, we present an analogous case with focus on the clinicopathological and imaging features as well as the associated differential diagnostic problems.In rare instances, CML patients on dasatinib treatment may experience lymph node enlargement reversible upon dasatinib discontinuation.24,5) at 1 year, the tolerance was suboptimal with recurrent episodes of maculopapular rash eruption and periorbital edema exacerbating the pre-existing allergic conjunctivitis of the patient. CML treatment was switched to dasatinib and 5 months later the patient consulted for evaluation of a non-tender left axillary mass of recent onset.We present a 40-year-old male CML patient on dasatinib who developed lymphadenopathy. The patient was diagnosed with BCR-ABL1 positive chronic stage CML (low risk group ELTS score) 2 years ago following a workup for fatigue and left upper quadrant abdominal discomfort. Although treatment with nilotinib was effective with documentation of complete hematologic remission at 3 months and complete molecular response  positron emission tomography/computed tomography (PET/CT) scan with above head to mid-thigh images acquired using a GE Discovery 710 TOF PET/CT scanner 60 minutes after the intravenous tracer administration; attenuation corrected PET images were reconstructed in the 3 main anatomical planes and fused with the corresponding CT sections. Highly increased tracer uptake (SUVmax 16.7) was documented in grossly enlarged left axillar and subclavian lymph nodes and moderate (SUVmax 4.2) in a small right axillar lymph node . TKI treatment with nilotinib was reinitiated with good tolerance.The hypermetabolic findings of the PET/CT being highly suggestive of lymphoma prompted a meticulous examination of the whole specimen. However, the aforementioned diagnosis of reactive lymphadenopathy was retained with features compatible with drug induced lymphadenopathy. These findings have been previously described in patients treated with dasatinib or other drugs, specifically anticonvulsants; dasatinib was suspected as the etiological factor and discontinued. The patient was followed-up every 2 weeks for 2 months. The patient maintained hematologic remission and the lymphadenopathy subsided 2 weeks after dasatinib withdrawal and was clinically undetectable at 4 weeks. Nine weeks after treatment interruption a new PET/CT scan was performed documenting an almost complete metabolic normalization Fig. B. Of not3 but patients on dasatinib treatment may present lymph node enlargement characterized by follicular hyperplasia, with or without PTGC morphology. To our knowledge, 17 cases of DAL have been described in the literature.,6 The mechanism of the follicular hyperplasia remains unknown. It has been assumed that TKIs involved in B-cell activation through the BCR and the Akt pathways could promote B-cell proliferation. However, in some cases, including ours, additional paracortex expansion due to a heterogeneous population of eosinophils, plasma cells, histiocytes and immunoblasts, some of which with characteristics of lymphocyte predominant (LP) and Hodgkin and Reed/Sternberg (HRS) cells are present.–8 Taken together, these histologic findings have been described in lymphadenopathy associated with anticonvulsant therapy, more commonly with phenytoin, as well as with other drugs, and represent a drug-induced lymphadenopathy. ,10The differential diagnosis in a CML patient with localized lymphadenopathy may present significant diagnostic challenges. Along with the typical workup, the exclusion of extramedullary blastic transformation of CML is warranted. Lymphoma has scarcely been described in the literature in CML patients,18F-FDG uptake by enlarged lymph nodes being the metabolic imaging hallmark of high-grade lymphomas, heightened the index of suspicion of lymphoma in our case. However, this PET appearance is far from lymphoma-specific as any kind of fast proliferating or reactive cells, like in the context of inflammation, can also concentrate 18F-FDG avidly. Overexpression of the cell membrane bound glucose transporter (GLUT) molecules family, is an important first step of this process. In particular, reactive B-cells in PTGCs, have been shown to display strong GLUT1 expression. This phenomenon, along with reactive follicular hyperplasia, harboring a variety of hypermetabolic, dividing cells, can lead to false positive 18F-FDG PET/CT results in both non-malignant and lymphoma conditions. Among dasatinib associated lymphadenopathy cases, 2 reported PET/CT results with hypermetabolic lymphadenopathy but further details were not provided.,5 It should be noted that hypermetabolic lymphadenopathy in PET scan has been described in drug induced hypersensitivity syndrome (DIHS) secondary to minocycline, but whether drug induced lymphadenopathy should be considered as part of the same spectrum of disease as DIHS is under debate, since their exact pathophysiological mechanisms are not well understood and no common underlying mechanism has yet been confirmed. In this case, the histopathological findings along with those of PET scan and the clinical course of the patient after drug cessation prompted us to consider the possibility that drug-induced lymphadenopathy may be implicated in DAL.The intense  However, in cases of viral- or drug- related PTGCs large cells may resemble LP or HRS cells making the differential diagnosis a challenge. In our case, immunoreactivity to T cell markers by few of the large cells, supported the possibility of immunoblastic reaction. In addition, there was no immunoreactivity to EMA antibody and the architecture and the surrounding to large cells microenvironment pointed to the correct diagnosis. Moreover, the coexistence of eosinophils and paracortical expansion in relation to clinical history favored the diagnosis of drug-induced lymphadenopathy. The resolution of lymphadenopathy following dasatinib discontinuation confirmed the diagnostic reasoning.The presence of PTGCs in our case, the etiology and pathogenesis of which remain unclear; along with immunoblastic proliferation may pose challenge to differential diagnosis mainly from Hodgkin lymphoma. PTGCs and nodular lymphocyte-predominant Hodgkin lymphoma share many histological features, with both entities presumed to be manifestations of an abnormal follicular center reaction and often correlated to each other. The presence of typical LP cells confirms the diagnosis of the lymphoma.418F-FDG PET/CT in this context should be cautious. On histological grounds, it may manifest with a spectrum of lesions including follicular and/or paracortical hyperplasia as observed in lymphadenopathy induced by other drugs. Further study is required to elucidate the pathogenesis of DAL.DAL may present as large mass. Interpretation of hypermetabolic lymphadenopathy detected by Written informed consent was obtained from the patient for publication of this case report.All authors made substantial contributions to the acquisition, analysis, and interpretation of data. All authors were involved in drafting and revising the manuscript and have approved the published version.


Acute respiratory failure (ARF) is a common and life-threatening medical emergency. Continuous positive airway pressure (CPAP) is a potentially beneficial alternative treatment; however, it is uncertain whether this could improve important outcomes in NHS ambulance services. The ACUTE study aimed to assess the feasibility of a large-scale pragmatic trial of pre-hospital CPAP.The study was a pilot randomised controlled trial of the O-Two system CPAP mask versus standard oxygen therapy, with concealed allocation in identical sealed boxes. Feasibility objectives estimated the incidence of eligible patients; the proportion recruited and allocated to treatment appropriately; adherence to allocated treatment; and retention and data completeness. The primary clinical endpoint was 30-day mortality. Ancillary studies included an ARF incidence study, ARF diagnostic agreement study, clinician perceptions of CPAP mixed methods study and investigation of allocation concealment.Over 12 months, 77 patients were enrolled (target 120). CPAP was fully delivered in 74% (target 75%). There were no major protocol violations/non-compliances. Full data were available for all key outcomes (targets ≥ 90%). Thirty-day mortality was 27.3%. Of deceased patients, 14/21 (68%) either did not have a respiratory condition or had ceiling of treatment decisions implemented excluding hospital NIV and critical care.The ACUTE trial recruitment rate was below the target rate and feasibility was not demonstrated. Identification of patients who might benefit from pre-hospital CPAP was challenging. It appeared difficult to exclude conditions where CPAP would not work, or might be harmful, and to select appropriate patients where there was a meaningful chance of success, or where the potential advantages of pre-hospital CPAP would outweigh the burdens of more advanced and aggressive treatment. The limited compliance with CPAP, and the difficulty in identifying patients who could benefit from CPAP, indicate that pre-hospital CPAP is unlikely to materially reduce mortality. A definitive effectiveness trial of CPAP in the NHS is therefore not recommended.


Graves’ hyperthyroidism is characterized by the presence of autoantibodies that stimulate the thyroid-stimulating hormone receptor (TSHR), resulting in uncontrolled secretion of excessive thyroid hormone. Conventional treatments, including antithyroid medication, radioiodine, or surgery have remained largely unchanged for the past 70 years and either lack efficacy for many patients, or result in lifelong thyroid hormone replacement therapy, in the case of the latter 2 options. The demand for new therapeutic options, combined with greater insight into basic immunobiology, has led to the emergence of novel approaches to treat Graves’ hyperthyroidism. The current therapies under investigation include biologics, small molecules, and peptide immunomodulation. There is a growing focus on TSHR-specific treatment modalities, which carry the advantage of eliciting a specific, targeted approach, with the aim of avoiding disruption of the functioning immune system. These therapies present a new opportunity to supersede the inadequate treatments currently available for some Graves’ patients, offering hope of successful restoration of euthyroidism without the need for ongoing therapy. Several of these therapeutic options have the potential to translate into clinical practice in the near future. This review provides a comprehensive summary of the recent advances and various stages of development of the novel therapeutic approaches to treat Graves’ hyperthyroidism.  The conventional therapeutic options for Graves’ disease have not improved over the past 70 years, despite substantial unmet clinical need and a significant lack of efficacy for many patients.The demand for innovative therapeutic options has led to the emergence of novel approaches in the treatment of Graves’ hyperthyroidism, including biologic, small molecule, and peptide immunomodulation, several of which have translational potential into clinical practice in the near future.Targeting the direct cause of hyperthyroidism in Graves’ disease using TSHR-specific modalities, has a clear advantage in providing a targeted approach while avoiding disruption of a functioning immune system.The introduction of novel therapeutics may lead to the restoration of a euthyroid state without the requirement for ongoing therapy, but the potential risk of immunocompromise and cost implications needs careful consideration.The therapeutic options available for patients with Graves’ hyperthyroidism have remained largely unchanged for the past 70 years, despite the current treatments having either limited efficacy or significant drawbacks. The classical approach to treating Graves’ hyperthyroidism involves the administration of antithyroid drugs to block thyroid hormone synthesis, or alternatively, destruction or removal of the thyroid by radioiodine or surgery . UnfortuGraves’ hyperthyroidism is an autoimmune condition that arises as a result of the loss of immunological tolerance to the thyroid-stimulating hormone receptor (TSHR) . ElevateHistologically, the thyroid gland in Graves’ hyperthyroidism demonstrates diffuse follicular cell hyperplasia with varying extents of lymphocytic infiltration into the thyroid stroma . These iThe TSHR is also found in orbital fibroblasts and the upper dermis where binding of TRAbs results in a proliferative response that contributes to the extrathyroidal signs seen in Graves’ hyperthyroidism, GO, and pretibial myxedema . ClinicaAlthough the specific immune mechanisms driving Graves’ hyperthyroidism remain unknown, a greater understanding of the interactions between B and T lymphocytes and the autoantigen-antibody complex has enabled progressive insights into the immunopathology underpinning Graves’ hyperthyroidism . The rolThe described immunological insights have led to the emergence of several novel therapeutic options, most of which remain under investigation in clinical or preclinical studies . In addiAs a humorally driven condition, the essential role of B cells in Graves’ hyperthyroidism provides a logical therapeutic target for immunomodulatory treatment. The B-cell depleting therapy rituximab (RTX) has been used for more than 20 years to treat lymphoproliferative malignancies, such as lymphoma, with increasing use over the past decade to treat autoimmune disease. Although the anti-CD20 monoclonal antibody RTX is the most widely studied of the B-cell therapies, the precise mechanisms by which RTX produces a beneficial effect remain uncertain. Direct apoptosis, complement-mediated cytotoxicity, and antibody-dependent cellular cytotoxicity all appear to contribute to RTX efficacy in depleting B cells . The CD2RTX was originally reported to have efficacy in controlling rheumatoid arthritis, with subsequent studies in myasthenia gravis (MG), anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, systemic lupus erythematosus (SLE) and multiple sclerosis . SeveralSome, but not all, studies have shown a reduction in serum TRAb antibody levels following RTX therapy , 15, 31,Most patients will have complete (>99%) B-cell depletion following a single 500mg dose of RTX, and this persists for 5 to 20 months before recovery of the peripheral B-cell compartment , with reAdverse effects have been reported with RTX, the most frequent of which is a mild infusion reaction including throat itching and nasal congestion, which resolves on slowing the infusion with or without the administration of hydrocortisone. There have also been reports of articular and gastrointestinal symptoms, specifically colitis, related to circulating immune complexes following RTX . The rarCD40, a tumor necrosis factor (TNF) family receptor found on thyrocytes and antigen-presenting cells, including B cells, has a primary role in coordinating effective antigen presentation . Its ligCD40 gene have been associated with several autoimmune diseases, including Graves’ hyperthyroidism, where they appear to influence thyroid antibody production and provide a predictive marker of relapse  targets the CD40–CD154 co-stimulatory pathway, resulting in attenuation of the B-cell activation signal . IscalimIn a recent open-label study, 15 adult patients with untreated Graves’ hyperthyroidism were given 5 doses of intravenous iscalimab over a 12-week period . During Iscalimab was found to be safe and well-tolerated, with no serious treatment-related adverse events reported. Similar to RTX, iscalimab is an immunosuppressive therapy and therefore risk of infection is always a concern. CD40 is expressed on vascular endothelium and platelets, so thromboembolic complications have been highlighted as another hypothetical risk—although this was not observed in the Graves’ disease patients treated. Unfortunately, after the 24-week follow-up period, 4 of the 7 “responders” relapsed and 3 of these patients required low-dose antithyroid medication.Targeted blockade of the CD40–CD154 interaction shows promising results in Graves’ hyperthyroidism, at least in the short-term and in those with lower TRAb levels (<20 IU/L). The fact that CD40 receptors are also present on orbital fibroblasts suggests the potential for a beneficial effect on GO as well. Indeed, 2 patients with GO whose hyperthyroidism responded to iscalimab also had improvement of eye symptoms and signs . An RCT The long half-life associated with IgG antibodies, such as TRAbs, is attributed to the neonatal immunoglobulin Fc receptor (FcRn), which recycles endocytosed IgG antibody by binding it in the acidic conditions of the lysosome and recycling it to the cell membrane for release back into the circulation . FcRn-de1-derived Fc fragment, while rozanolixizumab is a humanized, IgG4 anti-FcRn monoclonal antibody, both of which block FcRn-IgG interactions, thereby inhibiting IgG recycling and accelerating the removal of pathogenic IgG autoantibodies from the circulation , and pemphigus vulgaris. In a phase 2 RCT of efgartigimod in 12 patients with generalized MG, a rapid decrease of up to 70% in total IgG and anti-acetylcholine receptor autoantibody levels was demonstrated, alongside a rapid and sustained clinical improvement in 75% of those treated, compared with 25% of the placebo group . RozanolOther potential Fc and FcγR-targeting therapeutics for autoimmune disease are in various phases of development from preclinical to phase 2 studies . AlthougB-cell activating factor (BAFF) is a member of the TNF family of cytokines and has an essential role in B-lymphocyte activation, differentiation, and survival. Elevated circulating BAFF levels have been found in patients with several autoimmune conditions, including active Graves’ hyperthyroidism, where both degree of elevation of thyroid hormones and TRAb concentrations have been demonstrated to correlate with serum BAFF levels . GeneticThe BAFF monoclonal antibody belimumab binds to and antagonizes the biological activity of soluble BAFF. Blocking the interaction of BAFF with its receptor negatively effects B-cell proliferation, indirectly decreasing B-cell survival and reducing production of autoantibodies . The incEfficacy of belimumab alone, or in combination with RTX, has been demonstrated in SLE, with significant improvements in long-term organ damage , 79. ConAs discussed above, the isolated use of a B-cell depleting therapy  to treat Graves’ hyperthyroidism may lack efficacy due to the persistence of long-lived plasma cells and residual memory cells. Furthermore, following B-cell depletion there is a surge in plasma BAFF concentration. Concurrent targeting of both the homeostatic factors that are responsible for B-cell proliferation and differentiation, along with a B-cell depleting therapy may enhance the effect on tissue-resident autoreactive B cells.As the direct cause of hyperthyroidism in Graves’ disease is stimulation of the TSHR, several groups have been developing approaches that directly prevent TSHR signaling, either through small molecules or by using antibodies that block receptor activation. Furthermore, TSHR peptides are being investigated to see if they may have long-lasting immunomodulatory properties. A key advantage of this strategy when compared to the immunomodulatory strategies described above is their more specific, targeted approach which—from a theoretical standpoint—is unlikely to have a deleterious impact on the patient’s ability to fight infection.Small molecule agonists and antagonists have the potential to directly stimulate or inhibit TSHR signaling that could lead to highly potent therapies for thyroid dysfunction. A series of compounds that inhibit TSHR function as inverse agonists  have been developed; the best studied compound has been termed ANTAG-3 , 18. TheOccasional patients with GO may present with active inflammatory eye disease in the presence of hypothyroidism and high titers of TRAb antibodies, which contain a proportion of TSHR-blocking antibodies. Sanders et al were able to clone a monoclonal TSHR-blocking antibody, termed K1-70 from such a patient . In ratsConventional approaches to combating autoimmune disease involve using drugs with broad immunosuppressive effects and hence the potential side effect of susceptibility to infection. An alternative approach that has been successfully used to treat allergic disease is to desensitize the immune response to the specific allergen (immune stimulant) . The priThe described novel therapeutic approaches are not without potential limitations. As well as cost implications and the potential risk of immunocompromise with the non–TSHR-specific therapies, it remains unclear whether they will ameliorate the long-term risk of hypothyroidism, reduce goiter, or indeed prevent a late relapse of Graves’ hyperthyroidism. It seems likely that patients with Graves’ hyperthyroidism will continue to be at risk for thyroid dysfunction after cessation of therapy, necessitating annual biochemical monitoring.Conventional treatment of Graves’ hyperthyroidism with surgery, radioiodine, or antithyroid drugs has not substantially changed since the late 1940s. An abundance of new therapeutic approaches, involving biologic, small molecule, and peptide immunomodulation are currently at different stages of development and several will translate into the clinic over the next few years. These therapies may render destructive radioiodine thyroid ablation and thyroidectomy obsolete treatments of historical interest, although the advantages of restoring a euthyroid state without the need for ongoing therapy will need to be balanced against potential risks such as immunocompromise. Future studies need to focus on understanding the most effective combinations of the conventional medical and surgical therapies with these recently introduced options.


Although vascular alterations in solid tumor malignancies are known to decrease therapeutic delivery, the effects of leukemia-induced bone marrow vasculature (BMV) alterations on therapeutic delivery are not well known. Additionally, functional quantitative measurements of the leukemic BMV during chemotherapy and radiation therapy are limited, largely due to a lack of high-resolution imaging techniques available preclinically. This study develops a murine model using compartmental modeling for quantitative multiphoton microscopy (QMPM) to characterize the malignant BMV before and during treatment.trans), fractional extracellular extravascular space (Vcc), and vascular permeability parameters, as well as functional single-vessel characteristics. In response to leukemia-induced BMV alterations, the effects of 2 to 4 Gy low-dose radiation therapy (LDRT) on the BMV, drug delivery, and mouse survival were assessed post-treatment to determine whether neoadjuvant LDRT before chemotherapy improves treatment outcome.Using QMPM, live time-lapsed images of dextran leakage from the local BMV to the surrounding bone marrow of mice bearing acute lymphoblastic leukemia (ALL) were taken and fit to a 2-compartment model to measure the transfer rate (Ktrans, increased Vec, and increased permeability compared with healthy mice. Angiogenesis, decreased single-vessel perfusion, and decreased vessel diameter were observed. BMV alterations resulted in disease-dependent reductions in cellular uptake of Hoechst dye. LDRT to mice bearing ALL dilated BMV, increased single-vessel perfusion, and increased daunorubicin uptake by ALL cells. Consequently, LDRT administered to mice before receiving nilotinib significantly increased survival compared with mice receiving LDRT after nilotinib, demonstrating the importance of LDRT conditioning before therapeutic administration.Mice bearing ALL had significantly altered KThe developed QMPM enables single-platform assessments of the pharmacokinetics of fluorescent agents and characterization of the BMV. Initial results suggest BMV alterations after neoadjuvant LDRT may contribute to enhanced drug delivery and increased treatment efficacy for ALL. The developed QMPM enables observations of the BMV for use in ALL treatment optimization. Malignancy-related vessel remodeling can result in changes in BMV functional characteristics for some hematologic malignancies.11 To effectively optimize therapeutic delivery, both the pharmacokinetics of the therapeutics and the functional vascular characteristics  must be understood. Attempts to optimize the BMV on the basis of pharmacokinetics alone may result in further vascular dysfunction.12 Alternatively, optimization based on solely the BMV characteristics does not account for the wide variety of sizes and types of therapeutics (target or nontargeted).13 Therefore, effective methods that simultaneously observe vascular functional characteristics and therapeutic pharmacokinetics are essential to condition the vasculature in combination therapy.The optimization of therapeutic delivery is challenging because of the heterogeneity of malignant vasculature and the complex response of the vasculature to treatment.15 limitations are involved. First, such methods do not have the resolution required to identify the features of capillary-sized vessels, making observations of the underlying vascular functional characteristics difficult or impossible.16 Second, the resolution of such modalities often makes it challenging to observe tracer uptake in the bone marrow of a mouse model, limiting usefulness in a preclinical setting for hematologic applications. Alternatively, microscopic imaging modalities can identify the vascular functional characteristics but have not been used thoroughly to quantitatively assess therapeutic pharmacokinetics using kinetic compartmental modeling.Although methods to assess therapeutic pharmacokinetics  exist,18; (2) it has similar proportions of hematopoietic stem cells and similar hematopoietic reconstitution potential compared with bone marrow regions that are considered the primary sites of hematopoiesis19; and (3) it is a flat superficial bone requiring minimal invasive surgery before live imaging. For QMPM, time-lapsed images of fluorescent dextran leakage from the vascular lumen to the interstitial tissue were fit to a compartmental kinetic model to quantitatively assess (1) the kinetic transfer rate of dextran to the tissue space (Ktrans), (2) the fractional extracellular extravascular space (Vcc), and (3) vascular permeability, while also monitoring functional BMV characteristics including single-vessel blood perfusion, functional vessel surface area, vascular morphology, and vascular density.To address the need for a single imaging platform capable of observing the BMV functional characteristics and pharmacokinetic alterations from LDRT, we developed and validated compartmental modeling for quantitative multiphoton microscopy (QMPM) time-lapsed imaging of the BMV in the calvarium of live mice bearing GFP+ Philadelphia chromosome B-cell acute lymphoblastic leukemia (ALL). The calvarium region was selected for QMPM because (1) it contains sinusoidal vessels similar to those in other hematopoietic bone marrow regionsAfter seeing changes in the BMV with the onset of ALL, we hypothesized that LDRT (2–4 Gy) may improve vascular function and morphology and reduce leukemic burden, leading to better uptake of therapeutics and enhanced efficacy when used as a neoadjuvant therapy before therapeutic treatment. The response of the BMV to LDRT in mice bearing ALL was assessed using 2 treatment types: (1) targeted LDRT to half of the calvarium and to the left femur (tLDRT) and (2) total body irradiation (TBI). tLDRT was used to only irradiate small volumes of interest, ensuring that the systemic blood perfusion of the cardiovascular system was not affected with treatment.20 Both agents are of similar size to many small-sized therapeutic agents used for the treatment of hematologic malignancies.8 In addition, the efficacy of nilotinib (MW: 529.52), a similar-sized therapeutic agent effective for the treatment of ALL,20 was administered before or after 4 Gy TBI to determine whether LDRT-related changes to the malignant bone marrow and BMV may have an impact on treatment outcome in combination therapy.To assess the uptake of therapeutics, cellular uptake of fluorescent agents in regions receiving tLDRT and matching regions outside the radiation field  were compared to assess therapeutic delivery. Assessments were made 2 days after tLDRT and 20 minutes after intravenous injection of Hoechst 33342 dye  and daunorubicin HCL , a chemotherapy regimen used in the treatment of ALL and other hematologic malignancies.6 GFP+BCR-ABL (p190Kd) expressing B-cell ALL cells,21 suspended in 200 μL of phosphate-buffered saline (PBS), through the tail vein. To test drug delivery, injection concentrations of 10 mg/kg and 40 mg/kg were used for fluorescent Hoechst 33342 dye  and fluorescent daunorubicin HCL , respectively. Mice were imaged or euthanized for tissue processing 20 minutes after tail vein injection of Hoechst or daunorubicin. Nilotinib  treatments for survival studies were given at 50 mg/kg using oral gavage. Further details can be found in the All procedures for animal experimentation were performed according to City of Hope guidelines and approved by the Institutional Animal Care and Use Committee. C57BL/6J mice  mice were injected with 1 × 1022 Imaging was performed with a Prairie Ultima multiphoton microscope . All imaging was performed at single-day time points, and mice were euthanized after imaging sessions. Vascular blood pool imaging was performed with intravenous injections of 20 μL of Qtracker 655 Vascular Label  added to 80 to 180 μL of phosphate-buffered saline. Second harmonic generation imaging was used to observe the collagen in the bone regions and identify pockets of calvarium marrow within the bone. Velocities of cells flowing through the BMV network were measured using QMPM and calculated in a similar fashion as in previous works.23 Static image processing and tiling of large image sets were performed using Fiji/ImageJ25 and CellProfiler (v.2.2.0).26For QMPM calvarium imaging, mice were anesthetized using isoflurane, and surgery was performed to remove the skin and periosteum above the cranium. A custom titanium head plate was affixed directly to the calvarium and inserted directly into a heated microscope stage for stability during imaging.trans, and Vec were measured by time-lapsed imaging of 150-kDa TRITC dextran  leakage through the BMV. Mice were intravenously catheterized and injected with 500 μg of TRITC dextran during image acquisition. To identify blood and tissue compartments and obtain time-curves of dextran fluorescent intensity, a series of automatic thresholds was calculated for time-lapsed images using the Otsu method27 . See 29 CT image processing, contouring, and visualization were performed with Vivoquant  software. Dose calculations were performed with the small animal RT planning system29 using a Monte Carlo dose engine.31Computed tomography (CT) images were administered and acquired with the Precision X-RAD SMART Plus/225cx small animal image-guided irradiation system .Mice were administered tLDRT treatments in a single fraction of 2 to 4 Gy soft tissue–equivalent dose to the left half of the calvarium and left femur. tLDRT treatments were used to assess drug delivery and disease burden by comparing treated regions  and abscopal regions . TBI single-fraction treatments of 2 to 4 Gy were administered at matching soft tissue doses to tLDRT treatments, enabling comparisons of the irradiated BMV in the left half of the calvarium for tLDRT treatments  and TBI treatments (systemic cardiovascular treatment). All imaging and tissue harvests were performed 40 to 48 hours postirradiation. See 32 Briefly, endothelial cells were quantified as CD45/TER119− CD31+ identified from crushed femur and tibia bone samples. Bones were incubated in collagenase to liberate the leukemic and bone marrow microenvironment cells from the bone surface. Measurements of endothelial cell counts, cellular uptake of Hoechst, cellular uptake of daunorubicin, and GFP+ ALL cell counts were performed in standard fashion. Complete details can be found in Endothelial cell analysis was performed according to Kumar et al.Well plate fluorescence readings of TRITC dextran concentration were read from supernatants taken from crushed single-femur cell suspensions after removal of cells with centrifugation. Further details are found in t test with a P value < .05 considered significant. Significance testing for survival data was calculated using the log-rank Mantel-Cox test. For linear correlation testing, the R2 coefficient of determination tests were used. All distribution error bars are displayed as the mean ± 1 standard error of the mean.All statistical testing was performed using Prism . Unless otherwise noted, significance testing between groups was performed using the Welch 2-sided Large QMPM 3-dimensional tiled images of separate mice showed steady progression of ALL disease burden in the calvarium from 2 to 14 days post-ALL injection . Degradaec  and damage vasculature, causing increased leakage.35 A dose of 4 Gy tLDRT significantly increased Vec and Ktrans in tLDRT-treated calvarium regions compared with regions in untreated healthy mice , indicating that the change in Vec may be due to other additional factors, such as bone degradation or high interstitial fluid pressure. At low-to-moderate ALL engraftment, Ktrans was significantly higher than in healthy control mice (P = .0488)   and 2K. = .0488) . A signiTime-lapsed QMPM imaging of daunorubicin was performed in a mouse bearing ALL and compared with dextran kinetics to ensure that QMPM-based, time-lapsed imaging can be used for fluorescent agents of varying sizes and pharmacokinetic uptake patterns. A clear distinction between vascular, tissue, and cellular compartments could be seen in daunorubicin images . Faster −2, P = .0017, P = .0032, P = .1744, P < .0001, 36 These data suggest that ALL transforms the endothelial subset away from a hematopoietic supporting niche in support of ALL.QMPM measurements of calvarium mean vessel density were higher in mice bearing ALL compared with those in healthy control mice . Control mice typically had small inner diameter vessels with rapid blood flow connected to slow flowing vessels with a large inner diameter. Alternatively, mice with moderate-to-high ALL burden displayed a more homogenous population of vessels in terms of both blood flow velocity and vessel diameter, suggesting that typical cellular extravasation, oxygen delivery, and drug delivery may be altered in mice bearing ALL  in the average inner diameter of vessels in the calvarium in mice bearing ALL compared with healthy control mice . Similar BMV changes were observed with 2 Gy TBI treatments, suggesting that systemic LDRT to the cardiovascular system does not largely affect blood flow and that functional vascular changes may be beneficial for a variety of systemic or targeted RT treatments.Next, the effects of LDRT on the BMV in mice bearing ALL were tested with the hypothesis that LDRT would be beneficial in (1) improving the function and morphology of the BMV, (2) enhancing therapeutic uptake, and (3) increasing treatment efficacy when used before the administration of therapeutics. tLDRT treatments altered the morphology and functionality of the BMV in mice bearing ALL 2 days post-treatment -E. Mice P = .1744). This suggests that cellular Hoechst uptake is inhibited by the onset of ALL but not reduced due to a change in total cell number. In 2 Gy tLDRT-treated regions compared with abscopal regions, increases in the percentage of ALL cells labeled positive for Hoechst  and daunorubicin  were found  and 17 days  after the start of treatment (P = .0036, To test the treatment efficacy of the therapeutic delivery enhancements after neoadjuvant LDRT, we treated mice with 4 Gy TBI in combination with nilotinib. TBI was chosen for treatment because it had similar effects on the BMV as tLDRT and it was more suitable as a treatment regimen. A 4 Gy dose was chosen because it exhibits increased cell killing effects compared with 2 Gy with similar enhancements in therapeutic delivery. A treatment schema is shown in reatment . A signitrans, increased Vec, reduced vascular perfusion, increased mean vessel density, and reduced mean vessel diameter. ALL-induced alterations reduce cellular drug uptake of therapeutics. As hypothesized, LDRT increased BMV perfusion and enhanced therapeutic delivery to ALL cells in the bone marrow. Consequently, 4 Gy TBI before the administration of nilotinib significantly increased the survival of mice bearing ALL compared with treatment with TBI after nilotinib administration. These results demonstrate the ability of QMPM to characterize and optimize vasculature function for combination treatments of hematopoietic malignancies.In this study, compartmental modeling and multiphoton microscopy were combined to create QMPM, a single-platform method to both measure BMV functional characteristics and assess the pharmacokinetics of fluorescent therapeutic analogs. Assessments of mice bearing ALL revealed increased permeability, altered Ktrans and Vec. Changes in the vascular transport of molecular agents occur early in the progression of malignant disease, making Vec and Ktrans useful as potential biomarkers and for assessing drug or oxygen delivery.38 Limited work has been done with microscopy to perform tissue compartmental modeling for the identification of vascular permeability.23 Moreover, to our knowledge there have been no successful attempts using time-lapsed microscopy data to perform compartment modeling and obtain both Ktrans and Vec for the local vasculature. Differences in tracer concentration between the local vasculature and arterial blood may be present in poorly perfused tissues.28 In such a case, QMPM images of the local vasculature will represent the local tracer transport rather than the typical transport from arterial blood to whole tissue. However, in the case of well-perfused tissue, as is likely in this work, arterial and local vascular blood concentration measurements will be relatively similar. QMPM can be used in many different tissue types with many fluorescent agents, making it applicable to a wide variety of biological applications, such as the delivery of nutrients, oxygen, and therapeutics to the bone marrow.QMPM has the ability to model the transport of therapeutic analogs in the local tissue and vascular compartments to obtain Ktrans is increased compared with that in healthy mice, likely because of increased BMV permeability, enabling faster dextran leakage through the endothelial wall. Increased Ktrans values in low to moderate ALL burden may indicate a therapeutic window of enhanced delivery for ALL antibody therapies, such as those targeting CD19 or CD22, which are similar in size to 150KDa dextran.40 However, differences in vascular transport mechanisms of dextran and therapeutic antibodies make further assessments of antibody delivery kinetics necessary.41 In contrast to 150kDa dextran, small molecular agents such as Hoechst dye and daunorubicin are able to easily cross the vessel wall of both healthy and ALL-burdened BMV. This results in decreased cellular uptake of small-sized therapeutics with the onset of ALL, due in part to reduced blood perfusion.At low to moderate leukemic burden, dextran-based K23 With ALL vascular remodeling, the structure of the healthy BMV is lost because of unchecked angiogenesis, creating semicollapsed, immature, permeable vessels. This results in poor delivery of therapeutic agents, oxygen, and nutrients through the BMV. Increased hypoxia in the bone marrow has been reported with multiple hematological malignancies and is likely due to similar vascular alterations as reported in this work.42 Additionally, early preservation of the healthy BMV by the inhibition of vascular remodeling in vivo has been shown to give a survival advantage for leukemic mice treated with chemotherapy, suggesting drug delivery is hampered by the onset of malignancy.5 A restoration of BMV perfusion and reduction of high disease burden are crucial to improve the delivery of therapeutics to the bone marrow.Previous reports have shown arterial and sinusoidal blood vessels to be linked with phenotypes observed in healthy mice. Small-diameter, fast-flow BMV correlates with arterial vessels, whereas large-diameter, slow-flow BMV correlates with sinusoidal vessels and venules for healthy mice.43 Our results demonstrate the ability of LDRT to actively alter the BMV and indicate a possible new treatment approach for hematologic malignancies. Although recently developed total marrow irradiation is capable of delivering highly conformal treatment to the bone marrow45 or to smaller regions of high disease burden,46 it is often used in conjunction with high-dose regimens to enhance cell killing.47 In contrast, LDRT can provide both cell killing and vascular modulation to enhance therapeutic delivery in desired regions.This study suggests a possible new role for LDRT in restoring the biophysical function of the malignant BMV for combination therapies. Neoadjuvant LDRT treatments to ALL-burdened mice enhance BMV perfusion, increase delivery of therapeutics, and improve treatment efficacy. Similar RT-induced vascular alterations in solid tumors have been reported and may stem from nitrous oxide–dependent vasodilation.48 and therapies designed for vascular normalization frequently lead to pathway resistance, making combination therapy with RT desirable.50 Vascular targeted therapeutics show synergistic effects after low- to moderate-dose RT in solid tumor models, allowing for reduction in drug delivery without compromising treatment efficacy.51 Immunotherapy has also shown synergy with LDRT in solid tumor models.52 Additionally, LDRT for hematopoietic malignancies can be explored with more traditional therapeutic agents that elicit off-target toxicity or have synergistic effects.54With the wide variety of therapeutics that can be used with LDRT, consideration should be given to select synergistic treatment approaches. LDRT treatments may be particularly effective for vasculature targeting therapeutics, in which delivery depends only on vascular blood perfusion to endothelial cells. Vascular disrupting agents frequently elicit off-target toxicity,Although QMPM is useful to understand therapeutic delivery and microscopic vascular changes in the bone marrow, it is limited to preclinical studies only. Noninvasive macroscopic imaging such as dynamic contrast-enhanced MRI will need to be used to investigate the effects of LDRT on therapeutic delivery and the BMV for clinical translation. However, the resolution of macroscopic imaging makes it unsuitable for observing single vessel characteristics. Histology will need to be performed to observe the morphology of the BMV. Additionally, neither histology nor macroscopic imaging modalities will be able to measure the functional single-vessel parameters that can be observed with QMPM. In the future, correlation of QMPM’s microscopic imaging with macroscopic imaging will help in better understanding and interpreting the underlying BMV changes during macroscopic imaging for clinical translation.LDRT-induced alterations to functional BMV characteristics are one example of the many possible applications for QMPM. Simultaneous measurements of both functional single-vessel BMV characteristics and therapeutic pharmacokinetics using QMPM are able to reveal and optimize oxygen, nutrient, and drug transport for many disease models and tissues. In the context of ALL, such measurements are crucial to understanding LDRT treatment-induced BMV alterations and their effects on treatment outcome. Future work exploring LDRT will investigate the effects of timing, dose, and dose fractionation on the treatment of hematologic malignancies. QMPM measurements will be essential in observing the BMV for LDRT-based combination therapies.mmc7mmc6mmc6figmmc5mmc5figmmc4mmc4figmmc3mmc3figmmc2mmc2figmmc1mmc1fig


The COVID-19 pandemic has created an unprecedented global health care crisis. COVID-19 patients are found to have increased thrombotic risk. Despite being on prophylactic anticoagulation, many develop serious arterial and venous thromboembolic events. Emerging reports indicate COVID-19 may be considered a novel risk factor for portal vein thrombosis. Although, intra-abdominal infections are identified as risk factors, clostridium difficile colitis has not been typically seen as a risk factor for PVT. We report a case of an elderly female with a recent diagnosis of COVID-19 and no prior history of cirrhosis or malignancy who presented with diarrhea due to clostridium difficile infection. She developed sudden onset severe abdominal pain during the course of hospitalization. Acute portal vein thrombosis was identified on CT imaging of the abdomen, and she improved well with therapeutic anticoagulation. Acute portal vein thrombosis usually results from a combination of local and systemic prothrombotic risk factors. The combination of local infection by clostridium difficile and COVID-19 coagulopathy led to development of portal vein thrombosis in our patient. To the best of our knowledge, this is the first case of portal vein thrombosis reported in a patient with clostridium difficile infection in the setting of COVID-19 coagulopathy. During the current pandemic, clinicians should strongly consider abdominal imaging in patients presenting with abdominal pain due to clostridium difficile infection in the setting of COVID-19 to rule out complications such as portal vein thrombosis. Early diagnosis and treatment of portal vein thrombosis prevent complications of portal hypertension and intestinal infarctions. About 18% of patients diagnosed with Coronavirus disease 2019 (COVID-19) present with gastrointestinal symptoms , 4. AcutA 66-year old female with a history of fibromyalgia, gastroesophageal reflux disorder, traumatic brain injury, anxiety, depression, hypertension, constipation, and acute blood loss anemia during recent spine surgery seven weeks ago was transferred to the emergency room (ER) from a rehabilitation facility for watery diarrhea of 10 days duration and altered mental status for one day. The patient was receiving physical therapy at the facility for the past five weeks following elective spine surgery involving posterior L3-S1 fusion for pseudoarthrosis. She received perioperative antibiotics. Her medications included duloxetine, lubiprostone, losartan, propranolol, and gabapentin.She was diagnosed with SARS-CoV2 infection via nasopharyngeal swab testing at her facility during a routine screening two weeks before her presentation. She remained asymptomatic from a respiratory standpoint; however, she developed severe diarrhea and was receiving supportive treatment until she deteriorated.In the ER, vitals were remarkable for hypotension with blood pressure 70/50 mmHg. Labs showed elevated INR, elevated creatinine, leukocytosis, and low serum bicarbonate see . The abdDue to a lack of improvement in watery diarrhea, the persistence of abdominal pain, and the development of mild abdominal distension, a repeat CT scan of the abdomen/pelvis was obtained on the sixth day of the hospital course. It showed acute portal vein thrombus involving the left branch, moderate ascites along with findings of persistent colitis Figures  and 3. HPortal vein thrombosis (PVT) is caused when there is a partial or complete occlusion of the portal vein by a thrombus. Patients present with sudden onset severe colicky abdominal pain. Acute PVT results from a combination of local and systemic prothrombotic risk factors . In adulIt is well established that COVID-19 is associated with an increased risk of arterial thrombosis and venous thromboembolism , 11. In Intra-abdominal infections are known risk factors for PVT. However, Clostridium difficile has not been recognized as a risk factor for PVT before. Although PVT in COVID-19 patients was mentioned in a few cases, the unique presentation of PVT in a COVID-19 patient with Clostridium difficile colitis has not been reported previously to our knowledge. Our case report adds valuable new information to the practicing clinicians. In view of the current COVID-19 pandemic, portal vein thrombosis should be considered in patients presenting with abdominal pain in the setting of diarrhea, especially in patients with additional local risk factors such as Clostridium difficile infection. Early diagnosis and treatment of PVT with vitamin K antagonists or direct oral anticoagulants has the potential to reduce morbidity and mortality . Noncirr


The issues of pyrethroid resistance and outdoor malaria parasite transmission have prompted the WHO to call for the development and adoption of viable alternative vector control methods. Larval source management is one of the core malaria vector interventions recommended by the Ministry of Health in many African countries, but it is rarely implemented due to concerns on its cost-effectiveness. New long-lasting microbial larvicide can be a promising cost-effective supplement to current vector control and elimination methods because microbial larvicide uses killing mechanisms different from pyrethroids and other chemical insecticides. It has been shown to be effective in reducing the overall vector abundance and thus both indoor and outdoor transmission. In our opinion, the long-lasting formulation can potentially reduce the cost of larvicide field application, and should be evaluated for its cost-effectiveness, resistance development, and impact on non-target organisms when integrating with other malaria vector control measures. In this opinion, we highlight that long-lasting microbial larvicide can be a potential cost-effective product that complements current front-line long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) programs for malaria control and elimination. Microbial larviciding targets immature mosquitoes, reduces both indoor and outdoor transmission and is not affected by vector resistance to synthetic insecticides. This control method is a shift from the conventional LLINs and IRS programs that mainly target indoor-biting and resting adult mosquitoes. There has been a massive scale-up of antimalarial interventions since 2000 including long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), and artemisinin-based combination therapy (ACT). These interventions have led to significant reductions in malaria morbidity and mortality . HoweverAnopheles gambiae, An. arabiensis, and An. funestus . La. La26]. Box 1. Tests of LLML efficacy and effective durationBacillus thuringiensis israelensis (Bti) strain BMP 144 (1% in weight) and Bacillus sphaericus (Bs) strain AML614 (6% in weight). This product is being marketed in the US for mosquito vector control.The microcosm and field tests were conducted from 2009 to 2012 to determine the impact of LLML on vector abundance . The forBti/Bs briquette was placed in a 200 L water tank filled with rainwater and the tank was covered with fine mesh. The water was tested monthly for a period of 6 months to determine the effective duration [In this experiment, a duration . The resBti/Bs treatment experiments, with 41 treatment and 38 control habitats in western Kenya. Results indicated that LLML reduced malaria vector pupal productivity by 100% in the first two months and then by 63.4%–90.2% for 3–5 months after application  and productive (defined as containing larvae and pupae) habitats were selected for Anopheles adult densities, a clustered-randomized six cluster trial was undertaken in three areas of western Kenya. The briquettes were applied in all larval breeding sites. Mosquito abundance indoor and outdoor was monitored weekly using the CDC miniature light traps. The application of LLML caused a 66–88% (average 80%) relative reduction in the indoor Anopheles density (Fig. Anopheles density (Fig. Bti: Bacillus thuringiensis israelensisBs: Bacillus sphaericusCDC: Centers for Disease Control and PreventionLLML: Long-lasting microbial larvicideTo test if LLML reduces indoor and outdoor ity Fig. a and a 4ity Fig. b from weBox 2. Impact of LLML on non-targeted organisms and vector larval populationsAnopheles vectors was pooled in treatment and control clusters. The study was conducted from December 2015 to December 2016.As part of the ongoing large-scale clustered-randomized field trial conducted in western Kenya , field cApplication of LLML had no impact on the abundances of all non-targeted organisms collected during the entire study period Fig. . In addiLLML: Long-lasting microbial larvicide.Significant reduction in vector larval population density has been observed by week two of post-intervention Fig. . There wBefore LLML can be implemented on a large scale, improvements must be made on both the LLML formulation and the field implementation techniques. Antonio-Nkondjio et al. has outlined some of the important guidelines for the implementation of larval control interventions . SeveralThe cost-effectiveness of LLML should also be evaluated in other places than African countries. So far, LLML has only been evaluated in Kenya, although the conventional formulation of the same microbial larvicides has been tested/used and cost has been evaluated in other African –40, 46 aBti/Bs resistance has not been documented in laboratory or in field Anopheles mosquitoes although it has been tested widely for controlling malaria vectors [Bti/Bs formulation with a short effective duration for mosquito control in the USA and Europe has not led to high resistance in mosquitoes [Bti/Bs resistance. On the other hand, Bti/Bs has multiple toxin components, rendering resistance difficult to evolve [Resistance is another concern for LLML. So far,  vectors , 40, 54.squitoes , 55. Howo evolve . NeverthLastly, safety is a major concern regarding field application of any insecticides regardless of chemical or biological . It is gLong-lasting microbial larviciding represents a promising new tool that complements the currently front-line LLIN and IRS programs. It targets both indoor and outdoor transmission and alleviates the problem of insecticide resistance. Given the progress made in microbial larvicide formulation in conjunction with our improved understanding of mosquito ecology, in our opinion, long-lasting microbial larviciding may be a cost-effective supplemental malaria control method.Are LLML suitable for different ecological settings? In other words, how does environmental factor impact the effectiveness of LLML? The effectiveness of LLML may depend on local larval ecology, i.e., is it possible that LLML is more effective in some habitat types than the others?Conventional formulation of microbial larvicide has been shown effective in some settings, but it is costly. Is LLML cost-effectiveness for scale-ups?So far, no Bti/Bs resistance in Anopheles mosquitoes has been reported after decades of application, potentially due to its multiple toxin components. However, with the persistent and chronic selection pressure from LLML exposure, will mosquitoes develop rapid resistance to Bti/Bs?It is generally believe that Bti/Bs has no toxicity to people, various tests revealed no detectable harm to non-target organisms. Will persistent exposure to LLML cause harm to other organisms in the aquatic habitats?Before we can scale-up the LLML program, we need better understanding on some outstanding questions as described below:


P < 0.0001); however, FAP scores were not different between conditions (P = 0.786). Compared to single-contact stimulation, vCS elicited higher FAP scores with lower TEED (P = 0.031). FAP and TEED were positively correlated in vCS  and single-contact stimulation . Therefore, vCS and single-contact stimulation improved ambulation similarly but vCS reduced the TEED and side-effects at higher amplitudes.Vertical current steering (vCS) divides current between multiple contacts, which reduces radial spread to fine-tune the electric field shape and improves neuroanatomical targeting. vCS may improve the variable responsiveness of Parkinsonian gait to conventional deep brain stimulation. We hypothesized that vCS elicits greater improvement in ambulation in Parkinson’s disease patients compared to conventional, single-contact stimulation. vCS was implemented with divisions of 70%/30% and 50%/50% and compared to single-contact stimulation with four therapeutic window amplitudes in current-controlled systems. Walking at a self-selected pace was evaluated in seven levodopa-responsive patients. Integrative measures of gait and stimulation parameters were assessed with the functional ambulation performance (FAP) score and total electrical energy delivered (TEED), respectively. A two-tailed Wilcoxon matched-pairs signed rank test assessed the effect of each stimulation condition on FAP and TEED and compared regression slopes; further, a two-tailed Spearman test identified correlations. vCS significantly lowered the TEED ( Thus, in present clinical practice, PD patients with severe postural and gait instabilities or levodopa (L-DOPA) resistant postural and gait instabilities are often excluded from receiving DBS4. DBS chronically transmits electrical pulses from a sub-dermally implanted impulse generator (IPG) in the upper chest to neural tissue through implanted electrodes to address disabling dyskinesia and motor fluctuations from long-term use of L-DOPA5. The varying response of Parkinsonian gait deficits to DBS may be attributed to extensive physiological integration of various networks necessary for gait facilitation, the influence of bradykinesia and rigidity on Parkinsonian gait deficits, and the bias of upper limb symptom improvement during contact localization and selection7. Gait deficits attributed to rigidity and bradykinesia such as reduced step length and gait velocity tend to respond to L-DOPA and subthalamic nucleus (STN)-DBS. However, L-DOPA responsive gait deficits still exhibit a more variable responsiveness to STN-DBS as compared to appendicular symptoms such as tremor and rigidity. For instance, stimulation spread into the zona incerta and/or fields of forel from STN targeted electrodes has been reported to induce gait akinesia with a paradoxical improvement of dyskinesia, rigidity, and tremor9. This outcome highlights the sensitivity of gait treatment to neuroanatomical targeting of the resultant stimulation.Deep brain stimulation (DBS) consistently relieves appendicular symptoms in Parkinson’s disease (PD) but its effect on Parkinsonian gait deficits remains more elusive and variable10. IPG replacements are associated with a higher infection rate compared to the initial implantation and inadequate symptom alleviation is common following replacements despite programming with previously successful stimulation parameters 12. The clinical outcome and power usage are predominantly influenced by the electrode geometry and stimulation parameter magnitudes10. Electrode geometry is determined by the designation of the cathode and anode; unipolar configurations assign the contact(s) and IPG as the cathode and anode, respectively, and bipolar configurations assign one contact and a different contact as the cathode and anode, respectively10. Conventionally, single-contact unipolar configurations are programmed first; followed by, double contact unipolar then bipolar configurations upon failure of the previous configuration13. Unipolar stimulation is associated with a lower therapeutic threshold; thus, symptom alleviation occurs at lower amplitudes, which is beneficial for reducing power consumption10. However, the generated electric field is relatively broad due to the large distance between the cathode and anode10. Current spreads radially and commonly induces side effects, which are more pronounced with poor localization but more likely at high amplitudes regardless of localization10. Bipolar stimulation is associated with a higher side effect threshold; thus, higher amplitudes may be administered before inducing side effects. Namely, bipolar and unipolar stimulation elicit similar clinical benefits at lower stimulation magnitudes but unipolar stimulation is associated with more side effects at higher magnitudes14. Accordingly, less current spread is associated with bipolar stimulation since charge distribution encompasses a smaller volume as the anode and cathode are relatively closer10. However, modelling studies demonstrated that bipolar configurations may be less selective than unipolar configurations because neural elements are activated at both the anode and cathode15.Three major goals of DBS programming are to 1) optimize symptom alleviation, 2) reduce side effects, and 3) limit IPG power consumption to reduce the battery drainage rate. Faster drainage of the IPG battery results in an earlier need for replacement, which may occur as early as three to five years post-implantation in non-rechargeable systems10. Lower magnitudes tend to reduce power consumption but may be insufficient for effective symptom alleviation. For instance, low frequency stimulation between 13-30 hertz (Hz) has been reported to worsen bradykinesia16. Overall, the current/voltage, frequency, pulse width, and impedance influencing the stimulation may be expressed as a single variable termed the total electrical energy delivered (TEED)18. TEED is directly related to the power consumption and IPG battery drainage rate; thus, it may allude to the practical efficacy of stimulation22. TEED was first introduced by Moro and colleagues; however, it was later modified by Koss and colleagues and reported as follows:Furthermore, an optimal combination of frequency, pulse width, and current/voltage magnitudes must address the clinical outcome and power consumption24. Charge delivery of VC devices may not match the programmed value, which renders frequent programming adjustments due to inadequate stimulation of the target13. This is particularly an issue for programming during the early post-operative period  when impedance fluctuations are common. Namely, oedema as a result of surgical implantation increases the electrode impedance while stimulation decreases impedance25. Accordingly, tedious programming prompted the development of current-controlled (CC) systems with multiple independent current control23. CC devices adjust voltage to account for impedance fluctuations to ensure that charge distribution is consistent and that current amplitudes match the programmed value23. Multiple independent current control allows for manipulation of current delivery from each contact as a separate entity, which allows for directional and vertical current steering (vCS)26. Directional current steering activates individual contact segments of the ring rather than stimulating in the ring-mode (across the entire electrode circumference) to fine-tune stimulation in the axial plane. Alternatively, vCS divides current between multiple contacts stimulating in the ring-mode along the dorsal-ventral axis of the electrode. vCS targets or avoids neural structures by shaping the stimulation field by manipulating the percentage of current that is distributed from an active contact, which is directly related to the electric field spread. Thus, contacts stimulating with a greater percentage will exhibit a broader spread. Accordingly, vCS serves as a systematic technique to fine-tune the stimulation field to improve the accuracy of neuroanatomical targeting and algorithms associating divisions to contact localizations may replace present, empirical approaches to DBS programming27.Until recently, DBS systems have been pre-dominantly voltage-controlled (VC), which inconsistently deliver charge due to impedance fluctuations at the contiguous border between the neural tissue and contact7. Furthermore, employing separate current division programs may independently alleviate appendicular and axial symptoms to feasibly overcome the bias towards evaluating upper limb symptom improvement during contact localization and selection. Therefore, vCS may potentially address the variable improvement of L-DOPA responsive gait deficits feasibly and systematically.vCS is a programming technique that may improve the clinical outcome by shaping the stimulation field. vCS reduces diffuse radial spread of the conventional spherical field emitted from a contact, which reduces side effects related to stimulation spread into non-intended neural structures. Additionally, systematically tailoring current divisions to contact localizations may improve the accuracy of targeting the array of interacting circuits that regulate gait28. Additionally, the first multicentre study reported that majority of participants initiated programming with single-contact stimulation but switched to vCS for optimal symptom improvement by the one year follow up29. Reduction of the efficacy threshold allows for symptom alleviation at lower amplitudes, which decreases power consumption and slows battery drainage. Overall, this suggests that vCS is an advantageous technique as it allows for feasible systematic programming, which may improve the clinical outcome more efficiently. Thus, the rationale was to assess the ability of vCS to fine-tune stimulation in order to minimize the ad-hoc programming conventionally used to improve L-DOPA responsive gait deficits. Namely, vCS may address impedance fluctuations during the early post-operative period and stimulation of un-intended neural structures that may elicit side effects or sub-optimal symptom alleviation. Aforementioned issues have been inconsistently addressed with unipolar and bipolar stimulation with conventional VC-DBS systems.A few studies have exhibited the effectiveness of utilizing vCS to treat PD; namely, the first case study found that vCS reduces the efficacy threshold and increases the side effect thresholdThe purpose of our investigation was to compare the effect of vCS and single-contact stimulation on Parkinsonian gait deficits in CC devices. We hypothesized that vCS elicits a greater improvement in ambulation among PD patients compared to conventional, single-contact stimulation. The two main objectives were to 1) assess the individual effect of vCS and single-contact stimulation on integrative measures of gait parameters—functional ambulation performance (FAP) score and stimulation parameters—TEED, and to 2) determine the correlation and compare the relationship between FAP and TEED with vCS and single-contact stimulation.P = 0.705) and across amplitude stages (P = 1.000) (Table P < 0.0001)      Fig. ; however86) Fig. . A posit34) Fig.  and vCS -5) Fig. . With th10. Notably, vCS has been demonstrated to increase the side effect threshold, allowing for stimulation at higher amplitudes without inducing side effects28. Thus, the higher side effect limit of vCS represents greater practicality since one consistently used strategy to address worsened symptom severity associated with disease progression is to increase the stimulation amplitude30.No differences in the UPDRS motor symptom scores between vCS and single-contact stimulation across stimulation amplitudes were found, which was expected as all administered amplitudes were within the participants’ therapeutic window (TW). Moreover, TEED was minimized when vCS was employed but no differential effect was found on the FAP score when comparing the stimulation types Fig. . Since n22. TEED is directly related to the drainage rate of the IPG battery; therefore, reduced TEED can help delay the need for surgical IPG re-implantation to replace the battery. Compared to the initial implantation, re-implantation has been reported to elicit a higher infection rate and often requires a greater frequency of programming changes to achieve symptom alleviation12. Allert and colleagues reported that 20% of patients experience sub-optimal symptom relief despite accurate hardware restoration and programming with parameters that provided alleviation before IPG replacement11. Further, TEED reduction associated with vCS suggests that it is not associated with the same concerns of battery drain as other novel techniques such as interleaving stimulation, which independently and alternately stimulates from two contacts with different voltages and pulse widths at the same frequency31. Similar to vCS, interleaving stimulation may be implemented when unipolar and bipolar configurations fail to improve symptoms31.Furthermore, the TEED by vCS was significantly lower than the average TEED by single-contact stimulation Fig. . vCS min13. Single-contact stimulation is associated with a greater radial spread that arises from a single part of the electrode; thus, at higher amplitudes a broader current distribution may spread into non-intended structures, which elicits side effects. Conversely, radial current spread is reduced with vCS since the electric field is more elongated as a result of current division between multiple contacts10. Accordingly, algorithms relating the current divisions at respective amplitudes to contact localizations may improve the efficacy of neuroanatomical targeting and efficiency of conventional programming, which is ad-hoc and laborious13. Moreover, the positive slope for the linear trend line between FAP and TEED was steeper with vCS and the mean of the slopes of each participant’s regression in the vCS stimulation condition was significantly greater , and lowering the TEED (reduction of power consumption). Thus, vCS widens the TW, which was demonstrated in the first case study of vCS; symptom alleviation without side-effect induction was elicited at the same amplitude that single-contact stimulation elicited dyskinesia with sub-optimal symptom alleviation25. Further, the findings are limited to acute DBS effects as the IPG was turned on and vCS investigations began approximately four- and six-weeks following implantation, respectively. During this period, there is a greater frequency of impedance fluctuations and dosing changes of anti-Parkinsonian medication25. Oedema is postulated to increase the electrode impedance while stimulation rapidly decreases the electrode impedance25. VC systems are particularly sensitive to impedance fluctuations since they deliver voltage that is directly proportional to the electrode impedance30. Therefore, investigations during the early post-operative period are valid because this is when vCS is hypothesized to be particularly favourable due to the devices’ ability to address impedance fluctuations by dynamically manipulating the voltage to ensure constant-current delivery and to divide current to fine-tune the shape of the stimulation23. However, potential confounding effects from dosing changes of medication taken outside of testing sessions must be considered. Accordingly, another limitation would be the inability to completely restrict participants to DBS therapy over the investigational weeks, particularly at the lower stimulation amplitudes, due to medical beneficence. Thus, observed results cannot be fully attributed to DBS despite the supervising Movement Disorders specialist titrating oral medication doses to consistently minimize confounding and the discontinuation of medication for at least 12 hours before testing32. Similarly, the washout period of 25 minutes may not be adequate as Cooper et al. 2013 found that an adequate washout period for STN-DBS varies across participants and is inversely related with disease duration33. Lastly, unequal divisions of 70%/30% modelled vCS but the counterpart (30%/70%) was not investigated; therefore, the more ventrally located contact was biased with a greater proportion of current. Accordingly, future studies should personalize current divisions to optimally address each patients’ symptoms in a larger sample.The primary limitation to this investigation was the integrated analysis of segmented and non-segmented contacts, which was performed due to the small sample. TEED values for segmented contacts were inherently smaller because current is divided equally between segments. Thus, segmented contacts stimulate with a lower current and exhibit a lower TEED because the current is squared in the TEED equation (equation 3). Future studies should separate segmented and non-segmented contacts; notably, this may require a substantial increase in the sample size since segmented electrodes are only segmented at two levels. Thus, it is common for patients with segmented electrodes to be stimulated with a combination of segmented and non-segmented contacts. Additionally, the present investigation modelled conventional stimulation with a CC device to facilitate a within-subjects design that was employed due to the small sample and heterogeneous disease phenotype of PD. Nevertheless, a valuable future investigation should compare vCS to VC, single-contact stimulation to more accurately represent conventional practices. Moreover, TEED values may not precisely reflect the administration of a setting since impedance measures were obtained from a single time point, at least nine weeks following implantation, and the literature suggests that impedance may decrease during stimulation32. An adequate motor symptom response to L-DOPA was confirmed at least one week prior to implantation of the DBS system during the pre-assessment. Exclusion criteria included: 1) history of brain surgery, 2) previous implantation of a cardiac pacemaker, 3) overall poor health, and 4) tendency to exhibit lack of compliance. Ultimately, 10 individuals provided informed, written consent prior to their inclusion in the study. However, one patient voluntarily withdrew due to adverse health concerns unrelated to the study, one patient was implanted with a DBS system unable to perform vCS, and one patient had an inadequate motor symptom response to L-DOPA. Overall, seven participants with PD were included in the analysis of this study.This study was approved by the Human Subjects Research Ethics Board (REB) at Western University (REB #108453); accordingly, the study was performed in accordance with ethical standards established in the 1964 Declaration of Helsinki and its later amendments. Inclusion criteria included: 1) diagnosis of idiopathic PD with L-DOPA responsive motor symptoms as assessed with the standard L-DOPA challenge test (at least 25%); equation (1): TM PC IPG and the CartesiaTM Directional electrodes or the VerciseTM IPG and associated electrodes . Local and general anaesthesia were utilized for electrode and IPG implantation, respectively. The dorsolateral STN was targeted with standard stereotactic coordinates—4.0 millimetres (mm) ventral, 2.0 mm posterior, and 12.0 mm lateral to the mid-commissural point34. Intraoperatively, a stereotactic Leksell frame mounted the head for computed tomography imaging and electrode implantation. Spike recordings confirmed the STN positioning in the dorsal to ventral axis from five microelectrodes temporarily implanted to create a central, anterior, posterior, medial, and lateral channel. The implantation trajectory of the therapeutic macroelectrode was determined by the microelectrode that exhibited the most extensive STN spike signature and elicited the greatest improvement of tremor and rigidity upon intraoperative stimulation.All DBS devices (two electrodes and IPG) were implanted on the same day by functional neurosurgeons at University Hospital, London Health Sciences Centre . Participants were implanted with the VerciseThe first TW review was performed one-month post-implantation and involved a unipolar assessment of all contacts to identify the contact in the left and right STN that provided the most extensive TW. The TW was defined by the maximum and minimum or the side effect and efficacy threshold, respectively. The former is defined as the highest amplitude that elicits symptom improvement just before side effect manifestation and the latter is the lowest amplitude that provides symptom improvement. The TW was determined by increasing the current by increments of 0.5 mA until side effect manifestation. Subsequently, the previous amplitude was programmed and increments of 0.1 mA confirmed the exact side effect threshold. Finger taps, upper limb rigidity, and rest tremor were assessed for improvement, and postural tremor was additionally assessed for some participants. Blurry vision, face pulling, dystonia, dysarthria, and any other uncomfortable, intolerable sensations were noted as side effects. The second TW review was performed two weeks later, which paired the initially identified contact with all other contacts to find the pair that elicited the widest TW. Thus, the pair of investigational contacts were not necessarily adjacent and were only selected based on the extent of the TW. During the second review, each contact of the pair was stimulated with 50% of the current. Notably, the IPG was permanently turned on following the initial review, and participants accustomed to stimulation of 0.5 mA outside testing sessions. The frequency (130 Hz) and pulse width (60 microseconds (μs)) were consistent throughout the entire investigation; however, one participant was stimulated with a pulse width of 90 μs because 60 μs was inadequate for symptom alleviation.vCS investigations began immediately after the second contact review, which was approximately six weeks following surgical implantation. vCS was modelled with current divisions of 50%/50% and 70%/30% between two contacts and compared to single-contact stimulation, which amounted to eight investigational settings Table . All setMDS-UPDRS, Part III (motor examination). Of note, symptom severity is directly related to the UPDRS score; thus, higher scores indicate greater symptom severity. Moreover, gait assessments were recorded using a 0.61 metre by 6.1 metre ZenoTM walkway embedded with pressure-sensitive sensors . Forward walking was assessed at a self-selected pace without shoes, assistive walking devices , or ambulatory aids 35. Participants arose from a seated position completing five continuous loops in a clockwise direction to allow for speed maintenance and recordings in a uniform direction—away from the starting position. Overall ambulation was assessed with the FAP score, which was extracted from the ProtoKinetics Movement Analysis Software . Gouelle (2014) summarized the calculation of the FAP score as the deduction of points from a maximal score of 100 for aberrations from a healthy gait such as abnormal step time and length, stride velocity, and base of support35. Notably, scores between 95 to 100 represent gait in a non-diseased adult population36. The UPDRS and gait assessments on the pressure sensitive walkway were administered during the pre-assessment and vCS sessions.The UPDRS assessed motor symptoms at every setting across all amplitude stages as supplementary clinical data as the UPDRS is the gold standard for assessing PD symptom severity. Thus, scores were used to determine if a setting was representative of a PD patient receiving STN-DBS within their TW. UPDRS scores did not include toe tapping, kinetic tremor of the hands, and constancy of rest tremor, which are part of the Movement Disorder Society (MDS)-UPDRS, Part III (motor examination); nevertheless, the rater was certified to administer the 32. The pre-assessment also followed the standard protocol; “OFF” assessments preceded ON assessments during which, participants took a suprathreshold dose compared to their regular morning dose 32. As the stimulation amplitude increased throughout the study, dose reductions for medication taken outside of testing hours were prescribed by the supervising clinician to titrate concurrent anti-Parkinsonian therapies. Of note, dopamine agonists were slowly tapered based on the clinician’s discretion to avoid dopamine agonist withdrawal37.Participants were permitted to take L-DOPA, amantadine (to treat dyskinesia), and dopamine agonists outside testing sessions if medications were discontinued for at least 12 hours prior to the initiation of the pre-assessment, programming sessions to determine the TW, and investigational setting testing sessions. Thus, anti-parkinsonian medication was never taken during testing sessions with exception of the “ON” assessment of the pre-assessment. Discontinuation for 12 hours was based on the standard for acute challenges, which allows patients to be tolerably functional with adequate drug eliminationTEED values were calculated for each contact for every setting among the four investigated amplitudes for each participant Table . Voltage39. The effect of vCS and single-contact stimulation on FAP and TEED independently was assessed using the two-tailed, Wilcoxon matched-pairs signed-rank test. Additionally, a Spearman, two-tailed, correlation was performed to identify the potential correlation between FAP and TEED. Lastly, the regression between FAP and TEED in both conditions was compared by using a two-tailed, Wilcoxon matched-pairs signed-rank test. Differences between means of the slopes of linear trend lines for each participant’s regression were assessed with the FAP score set as the dependent variable.Statistical analyses were performed by GraphPad Prism 6.00  and R  using an alpha criterion of ≤0.05. Analyses utilized GraphPad Prism unless specified. Outliers for UPDRS and FAP scores were identified through diagnosis of disabling dyskinesia or dystonia by the supervising clinician. FAP scores were subject to additional outlier identification through the interquartile range rule; the FAP score and associated TEED value were removed if the FAP score was below the difference of the first quartile and the interquartile range multiplied by 1.5 or above the sum of the third quartile and the interquartile range multiplied by 1.5. Of note, UPDRS scores did not undergo outlier identification with the interquartile range rule because it served as supplementary clinical data. All data sets had a sample of seven (N= 7) and normality was assessed using the Shapiro-Wilk test. Differences in UPDRS scores between the two stimulation conditions and across the amplitude stages were identified with the Skillings-Mack test and a Nemenyi post-hoc test was planned; the former and latter were performed using R packages “Skillings.Mack” and “PMCMRplus”, respectively. Namely, the Skillings-Mack test is a non-parametric test used in place of the Friedman test (similar to the parametric repeated measures ANOVA) when there is missing data, and the Nemenyi post-hoc test is a conservative test used to perform pair-wise comparisonsThis study was approved by the Human Subjects Research Ethics Board (REB) at Western University (REB #108453); accordingly, this study has been performed in accordance with the ethical standards established in the 1964 Declaration of Helsinki and its later amendments. Additionally, all study participants provided informed written consent prior to their inclusion in the study.


The unpredictability of thyroid cancer can be striking, as the disease may rapidly progress to death in some individuals. Herein, we reported a rare case of aggressive papillary thyroid cell carcinoma (PTC) in an elderly patient de-differentiated into squamous cell carcinoma (SCC).We describe a case of a 79-year-old Thai woman presented with hoarseness and neck mass for 2 months and she had been diagnosed with a 3-cm papillary thyroid carcinoma (PTC) in the right side of the thyroid gland. Later on PTC de-differentiated into SCC within 3 years after initial presentation.De-differentiation from papillary thyroid carcinoma to squamous cell carcinoma.The patient underwent a total thyroidectomy at the initial hospital and received high dose radioactive iodine (RAI) treatment at our hospital 1 month following the surgery and then was lost to follow-up. Two years later she came back with new development of right solid-cystic neck mass which was found to be recurrent PTC. A radical neck dissection was done and another high dose RAI treatment was given. However, she developed recurrent mass with tenderness at the site above previous solid cystic mass 6 months later. Re-exploration of the neck mass revealed an inflamed midline mass 2 cm with enlarged right lateral cervical lymph nodes.A histopathological examination of the midline neck mass showed poorly differentiated SCC with lymphatic invasion. The intermingling of two morphologically distinct tumors, a typical PTC and a poorly differentiated SCC, had been identified in 1 out of 14 excised cervical lymph nodes. The patient underwent external beam radiation without chemotherapy. She is still in stable condition at 18 months post-treatment.This case clearly demonstrated that SCC transformed from a pre-existing PTC. The clinician should consider a possible transformation of papillary thyroid cancer into more aggressive histological types in elderly patients who present with rapidly progressive clinical behavior. However, some patients could have long-term survival if the tumor did not transform into anaplastic thyroid cancer. However, the clinical behaviors of PTC vary and are difficult to predict in some patients especially in very elderly patients (>75 years). Previous studies demonstrated that initial surgery approach, tumor size, extra-thyroid invasion, lymph node metastases, and histological subtype are related to the cancer recurrence,,5 but reliable markers to predict prognosis and seek intervention at early stage for aggressive PTC patients are not available yet in routine clinical practice.Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with its incidence having doubled over the past 2 decades from a widespread use of imaging to detect subclinical thyroid nodules rather than clinically significant PTC. However, the occurrence of squamous cell carcinoma (SCC) inside thyroid gland is uncommon condition because the normal thyroid gland has no squamous epithelial cells. Once SCC has been found from fine needle aspiration results or pathological specimens of thyroid gland, primary SCC versus secondary SCC needs to be differentiated from their diverse clinical and biological behaviors. PTC associated with SCC in the same lesion is an extremely rare condition in which collision tumor from other sites needs to be excluded.,9 Herein, we reported a rare case of aggressive PTC in an elderly patient that de-differentiated into SCC in which the intermingling of 2 morphologically distinct tumors typical PTC and poorly differentiated SCC had been identified in only 1 out of 14 excised cervical lymph nodes. The patient is still in stable condition at 18 months post-diagnosis. An informed written consent was obtained from the patient for publication of this case report and accompanying images, which have been approved by the ethics committee of Theptarin Hospital, Bangkok, Thailand.De-differentiation from well-differentiated thyroid cancer into poorly differentiated thyroid cancer and/or anaplastic thyroid carcinoma is frequently reported with dismal prognosis (<1 year in most cases).2A 79-year-old Thai woman presented with hoarseness and neck mass. She had been diagnosed with a 3-cm PTC in right side of the thyroid gland 2 months earlier. Her medical conditions included hypertension and heart failure with preserved ejection fraction. The patient underwent a total thyroidectomy at the initial hospital and received a high dose radioactive iodine treatment (150 mCi) at our hospital 1 month following the surgery and then was lost to follow-up. Preoperative serum thyroid hormone levels were within normal range and thyroid auto-antibodies were negative. No details on initial thyroid surgery and pathological report had been obtained.Two years later, she came back again with new development of a 3-cm right solid-cystic neck mass which was found to be recurrent PTC based on aspiration cytology. There was no evidence of distant metastases and there were no other obvious lymph node metastases in the neck. Radical neck dissection was done at our hospital and the second high dose radioactive iodine treatment was given . Pathological report revealed classical type of PTC without angiolymphatic invasion in excised tumor. However, she developed recurrent mass with tenderness at the site above previous solid cystic mass 6 months later. Additional work-up by neck ultrasound showed heterogeneous echogenicity of solid lesion with multiple internal calcifications at right thyroid bed (size 2.1 × 1.6 × 2.8 cm) with adjacent complex multilocular cystic lesion with septations and enlarged cervical lymph node with absent fat hilum  without chemotherapy because of her advanced age and co-morbidities. Concerned over airway protection, a prophylactic tracheostomy was done. The latest surveillance imaging done at 18 months after the last surgery revealed evidence of recurrent disease in the neck but the patient decided on watchful waiting without further treatments. She is still in stable condition at 18 months after the last operation.3 De-differentiation into poorly differentiated cancer or anaplastic cancer occurred in the only minority of patients which causes loss of iodine uptake ability. From a histopathological point of view, our patient demonstrated an unexpected finding of the development of SCC inside the lymph node of PTC metastasis. The final pathological diagnosis of PTC de-differentiated into SCC is important because treatment and prognosis differ from those of pre-existing well-differentiated thyroid cancer.Patients with PTC generally have very good prognosis but risks of lifetime recurrence vary (up to 25%) depending on lymph node metastases at the time of initial surgery, size of tumor, histological subtypes, and adequacy of tumor resection.–14 Based on clinical course of our patient and an immunohistochemistry staining, our patient clearly demonstrated that SCC transformed from a pre-existing PTC. The only identified SCC in 1 from 14 excised cervical lymph nodes allowed us to reach the correct diagnosis of PTC associated with SCC. The diagnostic skills of pathologists are extremely important in treating clinicians to handle difficult thyroid cases. Poorly differentiated thyroid carcinomas are a subset of thyroid cancer which is an intermediate phase between well-differentiated thyroid cancer and anaplastic thyroid cancers (ATC). In the past decade, the discovery of genetic landscape of thyroid carcinogenesis and progression helped to deepen our understanding of the complex and diverse clinical and biological behaviors of differentiated thyroid cancer. The progression to more aggressive behavior may be predicted based on both histological subtypes and molecular signatures. Subtypes of PTC including the tall cell, columnar cell, diffuse sclerosing, and oncocytic variants tend to be more aggressive and have higher risks of recurrence and metastases. However, in contrast to the previous reports of tall cell variant of PTC de-differentiated into SCC,–19 our case demonstrated the classical type of PTC as a previously reported case of an elderly Korean patient. Therefore, the unpredictable nature of PTC especially in very elderly patients should be kept in mind and lifelong follow-up is required in all cases of thyroid cancers.Apart from the de-differentiation process, the differential diagnosis of SCC inside the thyroid gland included primary SCC, metastatic disease from head and neck cancer, invasion from local structures, and the development of spindle cell squamous carcinoma which is a subtype of anaplastic thyroid carcinoma. A study from Asian patients showed that coexistence of TERT and BRAF mutations was found in 13% of PTCs and was significantly associated with older age and advanced stage compared with the group negative for either mutation. Therefore, identification of these mutations in routine clinical practice might have a role to delineate high-risk patients and offer intervention at early stage for those aggressive PTC patients. A previous study showed that the presence of identical mutation of BRAF gene in both primary thyroid tumor and metastasis site might help to exclude tumor to tumor metastasis. Unfortunately, both TERT and BRAF mutational analysis did not perform in our case. Future studies should be done to clarify the molecular pathway involved in the de-differentiation process.A recent study revealed that PTCs harboring Telomerase (TERT) promotor mutation are more likely to transform into ATC.As the tumor cells de-differentiated, cancer becomes resistant to the traditional therapeutic strategies, and prognosis worsens significantly. Optimal management requires balance between the risk of recurrence and adverse effects from treatment. After having completed surgical resection, these patients should ideally be offered adjuvant radiotherapy or adjuvant chemoradiotherapy. However, there are no standard chemotherapy guidelines for SCC due to de-differentiation from its rarity. Due to advanced age and co-existence of co-morbidities, our patient decided on watchful waiting without further treatments after adjuvant radiation. In contrast to primary SCC or ATC, long-term survival can be expected for some patients with poorly differentiated thyroid cancer if they undergo locally curative surgery and do not have distant metastasis or an undifferentiated component in the tumors.In conclusion, clinicians should consider a possible transformation of PTC into more aggressive histological types in elderly patients who present with rapidly progressive clinical behavior. This case provides further evidence that classical subtype of PTC can transform into SCC and it is important to consider this possibility in the presence of SCC inside the thyroid gland. The de-differentiation process from well-differentiated thyroid cancer portends worse prognosis but longer survival might be obtained if a complete surgical resection could be achieved. Further research and more case series are needed to determine a novel targeted therapy based on molecular characterization of the tumor.The authors wish to thank Dr. Tinapa Himathongkam for excellent language editing and the staff of Theptarin Hospital for all their support and help.Conceptualization: Yotsapon Thewjitcharoen.Investigation: Yotsapon Thewjitcharoen, Sirinate Krittiyawong, Pairoj Junyangdikul, Auchai Kanchanapituk.Supervision: Auchai Kanchanapituk, Thep Himathongkam.Writing – original draft: Yotsapon Thewjitcharoen.Writing – review & editing: Sirinate Krittiyawong, Siriwan Butadej, Soontaree Nakasatien, Somsong Polchart.Yotsapon Thewjitcharoen orcid: 0000-0002-2317-4041.