Osteosarcoma is the most common primary malignant bone tumor in adults and is usually located in the long bones. Standard treatment consists of perioperative chemotherapy and radical surgical resection. Limb-sparing surgery using a variety of reconstructive techniques remains the gold standard.In our study, we retrospectively analyzed 90 adult patients operated at our institution between 2000 and 2017 for extremity osteosarcoma that underwent limb-sparing reconstruction with the megaprosthesis. Sixty-one patients underwent resection and reconstruction of the distal femur, 9 patients—proximal femur, 7 patients—proximal tibia, 5 patients—total femoral resection and reconstruction, 5 patients—proximal humeral resection, and 3 patients—other types of resection with endoprosthetic reconstruction. The median follow-up time was 41 months, median overall survival was 86 months (3–225 months), and progression-free survival was 81 months (1–86 months). Functional assessment was made on 48 out of 56 living patients, after endoprosthetic reconstruction. The assessment was made according to MSTS functional scale.In 14 cases (15%), the endoprosthesis had to be explanted, or amputation was performed for local recurrence or septic complication. Due to a mechanical failure of the implant, we had to perform a revision in 5 patients (5%). Eighteen out of 74 patients with endoprosthesis died of the disease. The median MSTS score was 84% (53–100%), and the best result of 85% was achieved in patients after distal femoral resection with endoprosthetic reconstruction.Careful planning of the treatment of patients with extremity osteosarcoma that is performed at the referral centers gives the possibility of long-term survival with a good and excellent functional result. Osteosarcoma is the most commonly diagnosed primary malignant bone tumor with bimodal age distribution with its peak attributed to puberty and the fifth decade of life. The incidence of osteosarcoma in population is 0.2 per 100.000, which is less than 1% of solid tumors [Despite the increased risk of local recurrence, LSS is currently considered the optimal treatment solution. Published research shows that patients undergoing such treatment are characterized by a better total survival rate as compared with patients after amputation, not to mention reducing physical disability and its psychological effects that occur in patients undergoing such mutilating procedure –5.Amputation is necessary only in 10–30% of patients with extensive involvement of surrounding soft tissues, vessels, or nerves, in poor general condition or after improper, earlier surgical treatment. It is caused mostly by delay in setting a proper diagnosis and treatment, which leads to more advanced disease at the time of diagnosis.Due to more extensive muscle and bone resection in osteosarcoma patients, their functional results are inferior as compared to patients treated for degenerative disease.Most papers which present the functional outcome of sarcoma patients after endoprosthetic reconstruction contain small non-homogenous groups of patients with a variety of diagnoses from metastatic lesions to the whole spectrum of primary bone tumors.Many of them present the results of patients in a wide range of ages, from children to older people. In the case of children, their functional results are usually better than adults due to their better adaptation and regeneration possibility, which helps in more effective rehabilitation.In most cases, the diagnosis in children is made earlier, and resection is not as extensive as in adults. The most worldwide adopted/approved scale to measure the functional outcome of patients after limb-sparing surgery is Musculoskeletal Tumor Society (MSTS) scoring scale. This scale can answer the question of how the patients benefit from a limb-sparing surgery and allow comparing the variety of reconstruction techniques.The Department of Soft Tissue/Bone Sarcoma and Melanoma in one of the few referral centers in the East and Central Europe dedicated to complex treatment of the whole spectrum of bone sarcomas. In our department, patients are treated according to standard procedures adopted from NCCN and ESMO recommendation.This study aimed to evaluate the functional results of adult patients treated for limb osteosarcoma in our referral center with megaprosthetic reconstruction.We had retrospectively analyzed the history of adult patients treated at our institution due to osteosarcoma in years 2000–2017 with the use of inclusion and exclusions criteria that are shown on Table Finally, we have qualified 90 patients that underwent LSS with megaprosthetic reconstruction.The indications for LSS in our group of patients were resectable osteosarcoma without neurovascular involvement and adequate soft tissue envelope for prosthetic coverage.Tumor resectability was assessed on MRI or CT imaging performed after the third course of preoperative chemotherapy. According to AJCC classification in patients in stage IV of the disease, additionally, all metastases must be resectable after the preoperative chemotherapy courses. The resection of metastases was arranged after completing perioperative chemotherapy courses.The chemotherapy was started after pathological diagnosis, and staging of osteosarcoma was confirmed in open or needle core biopsy of the tumor.Routinely, we administered three courses of preoperative chemotherapy based on cisplatin and doxorubicin. From 2017 in patients under 30 years of age, we added methotrexate to routine chemotherapy. After surgery, patients received between 3 and 6 courses of chemotherapy according to the same preoperative schedule.All patients were operated at our department by an experienced team of surgeons between 3rd and 4th weeks after the last course of neoadjuvant chemotherapy.All patients underwent resection of osteosarcoma followed by reconstruction with megaprothesis.In the first 18 patients treated with endoprosthetic reconstruction, we used custom-made implants. In the rest of patients, we implanted modular endoprosthetic tumor replacements (MUTARS by Implantcast GMbH Germany).The minimum follow-up was 12 months from the end of treatment; the median follow-up time was 41 months. The mean age in the whole group of patients was 34 years (18–68). Patient characteristics are presented in Table In Table All patients were followed-up in our outpatient clinic. At the time of their appointment, we assessed them against the following criteria: pain, function, acceptance of treatment, support, walking ability, and gait. Each evaluated category was rated from 0 to 6 points.Functional effect of treatment was assessed in alive patients with implanted endoprosthesis with the help of the MSTS score system: 35 patients  after distal femur resection and reconstruction, 4 patients  after proximal femur resection and reconstruction, 2 patients  after total femur resection, 5 patients  after proximal tibia resection, and 2 patients  after proximal humerus resection and reconstruction.Median overall survival (OS) in the whole group of patients was 86 months (range 3–225 months), and progression-free survival (PFS) was 81 months (range 1–86 months).During follow-up, 18 out of 74 patients died from the disease between 3 and 69 months after the surgery.Age, survival time in months, AJCC stage of patients, who died, is shown in Table From a group of 90 patients after endoprosthetic reconstruction in 14 cases (15%), the endoprosthesis was explanted or the patient was amputated for local recurrence or septic complication.We removed 9 of 61 (16%) distal femoral endoprosthesis, 3 of 5 (60%) proximal humerus, 1 (11%) proximal femur, and 1 of 7 (14%) proximal tibia endoprosthesis. Five patients (5%) had a revision for mechanical failure of the implant. During follow-up, 18 of 74 patients with endoprosthesis died of the disease.The prosthesis survival curve is presented in Fig. The results of MSTS evaluation are displayed in Figs. Twenty-four patients (49%) of all assessed patients achieved MSTS score better than 90% in the whole group and 20 patients (52%) in the group of patients after distal femur resection and reconstruction.Achieved results show that patients had an excellent functional outcome of treatment, and they could return to a normal life after completing very demanding treatment.The example of the reconstruction and functional results in a patient with osteosarcoma of the distal femur is shown in Fig. In our series, local recurrence (LR) occurred in 15 patients (16%), and its presence was related to poorer OS. In a group of patients with LR, metastases occurred in 13 patients (86%) and 13 patients (86%) died of the disease. Comparing to a group of 75 patients without LR who had much better results, in which metastases occurred only in 18 patients (24%) and 14 (18%) of them died of the disease  occurred in our series in 16 patients (17.7%), and its presence was related to poorer OS. Occurring LR is connected to more aggressive disease and a much worse survival rate. LR is observed much more often in patients after non-radical resection (R1). LR occurred in 50% of patients with non-radical (R1) resection comparing with only 8.5% LR in patients after R0 resection.The main goal of LSS is to avoid disability and to give the patient a chance to return to normal life after demanding and challenging treatment. In the lower limb, the minimal functional effect is to give the patient a chance to mobilize with support with preserved limb sensation.In the upper limb, the minimal functional effect is hand grasping ability with preserved sensation. When this minimum requirement cannot be achieved with reconstructive surgery, it should be considered to perform amputation with an external prosthetic device. This type of treatment could give patients a better comfort of life than preserving the non-functional limb.Functional results in patients treated at our department with megaprosthetic reconstruction can be considered good or very good.The best results were achieved in patients after resection and reconstruction of tumor located in the distal femur with an average MSTS score of 85%.More than half of patients from this group achieved MSTS score of 90% or higher, which means that they returned to normal life without disability seen.In the majority of patients, resection and proper reconstruction allowed them to return to work or even to play recreational sports. That aspect of treatment is crucial to patients, especially at young age, to whom working or playing sport is an essential component of life quality. The worst functional results were obtained in patients after total femoral and proximal tibial reconstruction. These poor results could be caused by a small number of patients who were evaluated with the tumor in this particular location.The other reason for poor results could be the fact that resection of a tumor located in the proximal tibia is connected with the necessity of resection and reconstruction of a patellar ligament in the proximal tibia resection and resection of muscles stabilizing the hip joint and pelvis in total femoral resection. Above can lead to delayed rehabilitation and irreversible disability.In the majority of patients, reconstruction in the upper extremity can preserve the hand and elbow function, which helps to maintain an acceptable quality of life.Well planned and performed combined treatment of adult patients with extremity osteosarcoma leads to long-term survival with good or excellent functional results in the majority of patients.The correct treatment is the most crucial, non-disease-related, and modified-able risk factor for obtaining patients with long-term survival.Limb-sparing surgery is a gold standard of surgical treatment, and long-term survival rates are favorable in that group of patients comparing to a group of patients after amputation.Majority of patients treated with resection and megaprosthetic reconstruction allowed them to return to normal life and work. It also reduces physical and mental disability.Obtaining the correct and quick pathological diagnosis as well as starting the correct treatment in the referral center with no delay is the key to achieving the best results in patients with osteosarcoma.Establishing accurate and early diagnosis could lead to a decreasing number of performing amputations and allowed to start treatment in patients with less advanced disease.It is highly recommended to establish the referral center network for treating patients with sarcomas, including bone sarcomas, to give optimal diagnosis and treatment procedures without delay and to reduce the cost of treatment. That referral units participating in international clinical trials allow patients to get the latest and experimental therapies. Our department is a member of European Reference Network (ERN) for rare adult solid cancers (EURACAN), which gives our patient the best treatment opportunities.


The anchoring of the SMMs at the surface with an assumed statistic distance between each other is not an easy task; nevertheless, in this work, we show a synthesis which allows for this in detail. The immobilization of the Mn12–stearate was demonstrated with the use of FTO glasses and spherical silica as substrates. Based on differential pulse anodic stripping voltammetry (DPASV) and transmission electron microscopy (TEM) observations, we proved the efficiency of the method proposed. We observed continuous decreasing the number of bonds, and afterward, decreasing in the number of immobilized molecules with an increasing the number of spacer units used for separation of the magnetic particles.Controlling the distribution of the Mn The current developments in surface chemistry provide excellent opportunities for creating new materials and exploring novel and unique phenomena that take place on the surfaces of substances. In that vein, the functionalization of a surface plays an important role, and there are a great variety of techniques and methods aimed at modification of surfaces of nanoscale structures . SurfaceFor the organic functionalization of semiconductors silicon surfaces, self-assembled monolayers and direct attachment of oxygen- and carbon-free chemical functions  can be uA glass surface can also be used as a substrate to produce functional materials. In this case, using zeolite microcrystals as model microbuilding blocks  opens up12–stearate single-molecule magnets (SMMs) to the surface of spherical silica, allows one to separate the individual molecular magnets.However, an essential issue of surface functionalization, which was not included in aforementioned techniques, is the possibility to separate the individual functionalities and to control not only distances between them but their interactions. Such a solution is possible to realize using spacer units , which g12–stearate single-molecule magnets onto a surface of two types of substrates. The first one is FTO covered glass. The conductive substrate allows for the investigation of the samples utilizing electrochemistry methods. The second one is spherical nanosilica, allowing for the direct observation the Mn12-stearate SMMs [Here we present a procedure that provides a possibility of controlling statistic distance between SMMs on a surface by using spacer units. Theoretically, it is possible not only to separate the SMMs but also to tune mean intermolecular distances between SMMs by adjusting the number of spacer molecules: more separator molecules with regard to anchoring units, means a larger distance between anchored functionalities. In this study, we experimentally demonstrate this idea for the case of SMMs. We propose anchoring the Mnate SMMs . As the 12–stearate immobilization mechanism was more complicated than we assumed. The finding presented in this work seems to be very important for controlling the distribution of single-molecule magnets on the surface. The procedure allows for obtaining separated SMM on the surface and tuning the statistic intermolecular distances. The ability to attach isolated magnetic molecules on the surface will enable us to directly investigate their properties as single molecules in contrast to their study or extrapolation of their properties using a bulk analysis. The use of this approach opens a wide possibility for synthesis of materials that can be used in molecular-based magnetic devices (such as data storage and spintronics devices) and in quantum computation.The concept is a combination of those described earlier by our team ,15. AfteAs mentioned before, we prepared samples in two forms: based on FTO-glass (1), and as a fine powder based on spherical silica (2). Both were prepared in an analogous way. The synthesis routes involve a few steps, as illustrated in FTO-COOMn12, while those based on spherical silica hereafter are called SILS-COOMn12. The synthesis of Mn12–stearate was performed according to the protocol described previously [Before the proper chemical processing, we prepared substrates: FTO glasses  were degreased by the use of mechanical washing with a regular detergent. Next, the substrates were soaked in a solution containing a mixture of concentrated HCl  and methanol (1:1 of the volume) for one hour. After this process, we washed the substrates with distilled water and dried them by flowing dry nitrogen. Spherical silica was synthesized as described in the literature , and useeviously ,18.Solution I) was prepared. It contained the mixture of butyronitrile triethoxysilane  and tetraethyl orthosilicate  in toluene. The total concentration of TEOS and BNTES was 10−2 mol/dm3 for both substrates, while their molar proportion determined the distances between functional units: BNTES created the anchoring group for the functionality, while TEOS was a precursor of the spacer unit. We applied various proportions of BNTES and TEOS—1:0, 1:3, 1:6, 1:9, 1:12, and 1:15—which corresponded to the number of spacer units for each one anchoring molecule. In order to distinguish the samples, we added the number of spacers after the name; for example SILS-COOMn12N0 means spherical silica-based material does not contain any separators (N0—only BNTES), while sample SILS-COOMn12N15 includes 15 spacers per one anchoring unit . Analogously, we had FTO-COOMn12N0 with no spacers and FTO-COOMn12N15 with 15 spacers.In Step I we grafted the substrates with the precursors of the anchoring units and spacer groups. To that end, the . To do this we treated samples with a solution of chlorotrimethylsilane (ClTMS) in toluene (2% of volume)—Solution II. Pre-functionalized FTO glasses were immersed in this solution and for 24 h at room temperature. After this process the samples were rinsed with toluene and acetone. After drying under vacuum for two hours samples were ready for further processing.Step III) the cyanopropyl units were transformed into propyl carbonic acid groups (constituting the anchors for Mn12-stearate molecules) by hydrolysis. To do this we soaked the samples in the concentrated hydrochloric acid (37%—Solution III) for at least 5 h. Next, we rinsed the samples several times with deionized water, and dried under vacuum for two hours.During the (12–stearate molecules (Step IV). To do this we immersed the substrates containing propyl carboxy units in the solution of Mn12–stearate in dichloromethane (0.1 g of Mn12–stearate in 50 mL of CH2Cl2—(Solution IV)). The procedure was carried out in a weighing dish and under ultrasonification for 20 h. After this we rinsed the samples by the solvent for a few times to remove residues of the doping agent, and dried them under vacuum for a few hours. The resulting material was meant to posses SMMs immobilized at the substrate’s surface via bounding by propyl carbonic acid anchoring units. Functionalized FTO glasses were stored in a refrigerator under an argon atmosphere.In the last step, we functionalized the anchoring units (carbonic acid) by Mnspherical silica. Firstly, the spherical silica was mixed with Solution I under reflux overnight . Next, we recovered the product by centrifugation and washed it with pure toluene. We repeated this procedure four times, and subsequently dried the resulting powder under vacuum.Similar steps were applied for Solution II under reflux, similarly to what is described above.Next, the silylation was carried out by mixing the pre-functionalized powder with Hydrolysis was carried out by concentrated hydrochloric acid. We performed the reaction under reflux (12 h). Subsequently, we centrifuged the resulting powder and washed it by water and acetone .12–stearate. For the functionalization of 0.3 g of silica, we applied 50 mL of the Solution IV . We mixed this suspension overnight at room temperature under an argon protective atmosphere. The resulting powders  were recovered by centrifugation, washed by dichloromethane five times, and dried under vacuum for 10 h. Samples were stored in an argon atmosphere in a refrigerator.Finally, we functionalized the silica containing carbonic acid anchoring units on the surface with MnWe assumed that by the variation of the number of spacers, it would be possible to control the distribution of the anchored single-molecule magnets. To check our assumption, we decided to carry out two crucial investigations: differential pulse anodic stripping voltammetry (DPASV) for the case of samples prepared on FTO-glasses and TEM microscopy for spherical silica-based materials. The DPASV is a susceptible technique allowing for quantitative estimation of the number of the bonds between substrate and SMMs ,20. TEM 12 in this case.The DPASV spectra  of the FTO-glass-based samples are shown in 12–stearate molecules as well. In our opinion, however, this is unlikely, since in such a case, we would expect the peak or rather peaks’ positions to be different than what is typical for the stripping of carbonic bonds. Of course, we cannot exclude such a possibility.The position of the DPASV peak suggested that we were dealing with chemically bonded specimens . The relThe relative amounts of bounds can be estimated by measuring the area under the DPASV peaks  see val. The com12–stearate molecules at the surface. Nevertheless, we can clearly observe, that for the samples SILS-COOMn12N0, SILSCOOMn12N3, and SILS-COOMn12N6 SMMs are very densely distributed on the surface—molecule by molecule. No difference in distribution can be distinguished. In our previous work, we maintained that Mn12–stearate molecules were bonded at the surface in an umbrella-like configuration [SILS-COOMn12N0 and SILS-COOMn12N3. Closer inspection of the TEM image of SILS-COOMn12N6 sample reveals one important detail, however. This sample shows Mn12–stearate molecules that are bounded in different orientations like those marked in the The TEM results are quite surprising. For all cases, one can see separate Mnguration . That isSILS-COOMn12N9, SILS-COOMn12N12, and SILS-COOMn12N15 as well. Moreover, for these samples, we can clearly see a decrease in the number of anchored Mn12–stearate molecules. Why then can we observe a reduction in the anchored SMMs’ density after applying at least six spacers per anchoring unit? This finding seems to be inconsistent with the DPASV results. The explanation can be found by analyzing the molecular structure of the Mn12 molecule [12–stearate to the surface. Concentrations of the spacers of below this value do not cause an increase in the number of anchored SMMs but an increase the number of bonds between Mn12 and the support. This conclusion is in agreement with DPASV results: when the molecule is anchored via four bonds, its detection involves the exchange of four electrons and only one for a single bond. Thus, we observe continuous diminishing in the area under the DPASV, while TEM imaging shows roughly the same number of immobilized magnetic molecules, up to the six spacers per anchoring unit.This orientation of the SMMs can be observed for the samples molecule . It has 12–stearate molecules did not lose their SMMs properties. Moreover, SQUID research revealed extremely interesting behaviors of the material . The description of the magnetic properties of the materials is very extensive and is presented in a pending article. Magnetic research did not prove the light shed on the connection between Mn12–stearate and silica support though. For this reason, the only information about the chemical bonding between SMMs and the substrate was provided by DPASV.Here we must remark that some other research was carried out to check the chemical compositions of our specimens: energy-dispersive X-ray spectroscopy , Raman spectroscopy , and SQUID magnetometry . Due to the reason of very low concentrations of the functional groups, EDX and Raman did not bring unambiguous results. The SQUID magnetometry proved that the Mn12–stearate, not diminishing the number of immobilized SMMs though. The reducing number of anchored magnetic molecules can be observed when the number of spacers is more than six per one anchoring molecule. Moreover, we can say that the samples SILS-COOMn12N6 or FTO-COOMn12N6 are optimal: they possess the largest possible numbers of immobilized SMMs with the smallest possible numbers of bonds. For the case of immobilization with Mn12 with more than a single anchoring point, the molecule is relatively rigid. Its mobility is limited. Therefore, the samples with six spacers contained the highest possible concentration of non-rigid, single-molecule magnets. This fact is reflected in the magnetic properties of such materials (work in preparation).Summing up both TEM and DPASV research, we observed that with the increase in the number of spacer units up to six per single anchoring molecule, we dealt with a decreasing of bonds between the support and Mn


Drosophila, that we called the F-P boundary. Lawrence et al. present no data from the Lepidoptera and while the data that they present from Drosophila melanogaster mitotic clones are intriguing and may be informative with respect to the timing of the activity of the A-P and F-P organizers, considerable ambiguity remains regarding how their data should be interpreted with respect to the proposed wing compartment boundaries. Thus, contrary to their claims, Lawrence et al. have failed to falsify the F-P boundary hypothesis. Additional studies employing mitotic clones labeled with easily detectable markers that do not affect cytoskeletal organization or rates of cell division such as GFP and RFP clones produced by G-Trace or Twin Spot Generator (TSG) may further clarify the number of compartment boundaries in Drosophila wings. At the same time, because Drosophila wings are diminutive and highly modified compared to other insects, we also urge great caution in making generalizations about insect wing development based exclusively on studies in Drosophila.Here we reply to the “Refutation” of Lawrence, Casal, de Cellis, and Morata, who critique our paper presenting evidence for an organizer and compartment boundary subdividing the widely recognized posterior wing compartment of butterflies and moths (Lepidoptera) and Sci. Rep. 9 (2019), 10.1038/s41598-019-42668-y.Replying to: Lawrence, P.A., Casal, J., de Celis, J., Morata, G. A refutation to ‘A new A-P compartment boundary and organizer in holometabolous insect wings’.  We also used FLP-FRT induced mitotic clones to identify a possible homologous compartment boundary in Drosophila2. The bulk of our paper2, part of a series exploring the evolution and development of wing patterns in Vanessa butterflies4, focuses on data from butterflies and moths including a statistical analysis of butterfly eyespot phenotypes, butterfly wing immunohistochemistry, and a review of clonal boundaries on the wings of lepidopteran mosaic gynandromorphs and mosaic homeotic mutants.Here we make a brief response to Lawrence et al.1 make only one substantive comment regarding our lepidopteran experiments: that the independent contrast analysis of Vanessa butterfly eyespot structure does not show a similar effect of the A-P compartment boundary to that shown by the F-P compartment boundary. They assume that the organizers associated the A-P and F-P boundaries should have similar effects on colour pattern development. However, there are many examples of serially homologous developmental features  that are organized and patterned differently during development. There is no reason to assume a priori that both boundary organizers should have the same effects on colour pattern, especially if they are active at different stages of wing development.Lawrence et al.1 is largely a critique of a single experiment: our analysis of Drosophila mitotic wing clones marked with yellow mutations2 and created using the FLP/FRT technique6. On the one hand, they describe how they created “genetically marked clones rather as Abbasi and Marcus have done”1, while on the other hand they find our results unconvincing because our clones differ in shape from clones marked with multiple wing hairs that they have created by mitotic crossover using X-rays, in some cases in a Minute background. Given that Minute mutants have profound effects on cell growth7, clones that involve these factors (either in the clone itself or in the surrounding tissue) will differ in size from clones where the rate of cell division has not been manipulated8. Similarly, the multiple wing hairs mutant phenotype is a consequence of cytoskeletal reorganization9 that can disrupt epidermal cell polarity10. Further, X-ray exposure can have a dramatic effect on the proliferation dynamics of cells in the imaginal wing disc11, by causing both cell death and compensatory proliferation of surviving cells12 with profound effects on wing clone characteristics11. Thus, given the extent to which Lawrence et al.1 manipulated fundamental cellular traits to produce their clones, it should be no surprise that our reported clone shapes and sizes are different2.Thus, all of the findings that we presented from the Lepidoptera remain unchallenged and so rather than a “Refutation”, Lawrence et al.1 also question our use of yellow as a lineage marker in Drosophila. The yellow gene product is a secreted peptide that appears to be an essential cofactor in the L-dopachrome tautomerase enzyme complex13 which converts the soluble substrate dopachrome into insoluble 5,6-dihydroxyindole (DHI)14, a melanin precursor15. In the extracellular matrix yellow protein is embedded in the developing cuticle and may function in part to anchor pigment molecules to the cuticle16, so for the most part the yellow gene product functions cell-autonomously in mosaics18. For this reason, yellow mutants have been used extensively to mark clones for genetic mosaic analysis20. Drosophila yellow mutations have discernable wing phenotypes in both bristles and trichomes17 and these phenotypes have been used successfully by other workers to label wing clones in D. melanogaster22 and in D. hydei (using mutants in the homologous D. hydei yellow gene)23. We first encountered yellow wing clones when P-element mobilization-induced mitotic recombinants marked with y1 appeared incidentally in the course of a mapping experiment involving over 90,000 flies24. We are genuinely puzzled by the objections of our critics1 to the use of yellow to mark mitotic clones, when they have endorsed and popularized its use as a cell-autonomous marker in other tissues and contexts27.Lawrence et al.1, the patches of marked cells that cross the F-P boundary could be explained by contiguity of 2 or more adjacent mitotic clones on either side of the boundary such that they resemble a single larger “clone”. While they claim that the presence of 2 or more fused clones is improbable, it has been shown that the 1000 R dose of radiation used by Lawrence et al.1 will produce 2 or more clones in ~65% of all Drosophila wings with induced mitotic clones11. Further, in a Minute- background each individual Minute + clone becomes proportionately larger8, while the overall size the wing remains the same28, increasing the likelihood that 2 nearby clones will fuse and appear as a single larger, uniformly marked “clone” that appears to cross a developmental compartment boundary.Regarding the data presented by Lawrence et al.1 appear to respect the A-P boundary, but apparently violate the F-P boundary suggests to us that there may be a difference in the timing of the creation of these boundaries relative to the timing of mitotic clone creation in their experiments, possibly with the A-P boundary being established earlier in development. Differences in timing may also explain the results of the twin spot experiments in Drosophila (where the number of clones can be determined precisely) summarized by Table 1 of Lawrence et al.1 If X-ray induced recombination creates mitotic clones before a developmental compartment is specified, cells marked by one of the two mutations used to mark the twin clones can appear on both sides of the compartment boundary. This is similar to observations of bilateral and some mosaic lepidopteran gynandromorphs where cells in both anterior and posterior wing compartments are included within the same clone and thus marked with the same phenotype30.The fact that the marked “clones” of Lawrence et al.1 have suggested that the cellular behavior that we link to a possible F-P boundary in Drosophila wings may be due to the presence of a documented secondary organizer apparently without a corresponding compartment boundary in the posterior compartment of the Drosophila wing and haltere imaginal discs associated with Dpp expression during the 3rd larval instar31. Whether this additional Dpp-dependent organizer actually does coincide with an F-P compartment boundary as we have proposed in Drosophila2, or alternatively whether it exists in both Drosophila and other insects independent of any compartment boundary31, or finally whether the apparent compartment boundary-independent state in Drosophila represents an evolutionary derivative of an boundary-associated organizer found in other insects with less derived wing structure and development as proposed by Lawrence et al.1 all remain to be determined. To date, transcriptomic profiling of the homologous posterior region of butterfly wing imaginal discs has not recovered dpp transcripts32, so even if Dpp is the relevant patterning organizer in Drosophila, this may not be a general mechanism shared by all insects. In our published model based on observed patterns of A-P eyespot individuation in butterflies2, we also suggested that the A-P organizing signal (Dpp) and the F-P organizing signal are likely to be different gene products with independent patterning effects on wing tissue.Lawrence et al.1 join us in calling for additional research on these very interesting aspects of insect wing development. However, Lawrence et al.1 have not convincingly falsified the Far-Posterior compartment and organizer hypothesis in Drosophila melanogaster, nor have they presented any relevant data to address that hypothesis in butterflies or other insects. The current disagreement concerning Drosophila wing compartment organization might be resolved by studies employing mitotic clones labeled with easily detectable markers that do not affect cytoskeletal organization or rates of cell division (such as GFP clones produced by G-Trace33). Experiments that produce twin clones in the wing labeled with fluorescent markers such as those produced by the Twin Spot Generator (TSG) method34 would be even more definitive. Yet, regardless of these experimental outcomes, we must be very cautious about generalizing these findings because Drosophila wings are atypical, highly modified, and reduced in size with greatly reduced venation and hindwings converted to halteres36, potentially causing a profound bias in our understanding of insect wing development. Therefore we encourage other researchers who use insects other than Drosophila to join us and investigate the formation of compartmental boundaries in their insect models, from which we can gain a more comprehensive view of insect wing development.In conclusion, we welcome all critical scrutiny of our work and its interpretation, and we are pleased that Lawrence


Facioscapulohumeral muscular dystrophy is a rare inherited neuromuscular disease with an estimated prevalence of 1/20,000 and France therefore harbors about 3000 FSHD patients. With research progress and the development of targeted therapies, patients’ identification through registries can facilitate and improve recruitment in clinical trials and studies.The French National Registry of FSHD patients was designed as a mixed model registry involving both patients and physicians, through self-report and clinical evaluation questionnaires respectively, to collect molecular and clinical data. Because of the limited number of patients, data quality is a major goal of the registry and various automatic data control features have been implemented in the bioinformatics system. In parallel, data are manually validated by molecular and clinical curators. Since its creation in 2013, data from 638 FSHD patients have been collected, representing about 21% of the French FSHD population. The mixed model strategy allowed to collect 59.1% of data from both patients and clinicians; 26 and 14.9% from respectively patients and clinicians only. With the identification of the FSHD1 and FSHD2 forms, specific questionnaires have been designed. Though FSHD2 patients are progressively included, FSHD1 patients still account for the majority (94.9%). The registry is compatible with the FAIR principles as data are Findable, Accessible and Interoperable. We thus used molecular standards and standardized clinical terms used by the FILNEMUS French network of reference centers for the diagnosis and follow-up of patients suffering from a rare neuromuscular disease. The implemented clinical terms mostly map to dictionaries and terminology systems such as SNOMED-CT (75% of terms), CTV3 (61.7%) and NCIt (53.3%). Because of the sensitive nature of data, they are not directly reusable and can only be accessed as aggregated data after evaluation and approval by the registry oversight committee.www.fshd.fr and various useful information, links, and documents, including a video, are available for patients and professionals.The French National Registry of FSHD patients belongs to a national effort to develop databases, which should now interact with other initiatives to build a European and/or an international FSHD virtual registry for the benefits of patients. It is accessible at The online version of this article (10.1186/s13023-018-0960-x) contains supplementary material, which is available to authorized users. With an estimated prevalence between 1/20,000 and 1/8,000 [FSHD is characterized by an important clinical variability including age of onset, disease progression, and presence or absence of multisystemic involvement . FSHD paSMCHD1 gene located on chromosome 18. Alternatively, pathogenic mutations in the DNMT3B gene have been described in association with FSHD2 [FSHD is a genetically heterogeneous disease with two distinct forms. FSHD type 1 (FSHD1) is the most common form with more than 90% of FSHD patients, while the FSHD type 2 (FSHD2) is the rarest form of the disease, affecting less than 5% of FSHD patients. FSHD1 is caused by the contraction to 10 repeats or less of the 3.3 Kb D4Z4 repeat units on chromosome 4. In contrast, FSHD2 is caused by mutations in the chromatin modifier th FSHD2 .To date, in France, molecular diagnosis is available only for the FSHD1 type; indeed, clinical FSHD patients with no D4Z4 contraction might be explored in a research context, and positive results are not fully integrated in the diagnosis process yet.http://www.treat-nmd.eu). They are either disease-centric in the United-Kingdom [With the recent interest from pharmaceutical industries to develop novel therapeutic solutions for FSHD patients, it is now mandatory to create national registries to facilitate fully characterized patients’ recruitment in clinical trials and better understand the natural history of the disease to identify key parameters to efficiently evaluate treatment benefits. Today various national registries have been or are being set up as reported in the TREAT-NMD website  with the 4th dimension package . The website contains a mix of static and dynamic pages created upon request, using JavaScript and Ajax functions (https://www.w3schools.com/). The web interface was developed using the HTML5 and CSS3 standards with the Bootstrap 3.1 package, thus allowing easy access to the website from multiple platforms .The FSHD French National Registry is accessible at The self-report questionnaire collects general data, a clinical self-assessment, genetic diagnosis results, and the patient’s disease history  the patients who can fill self-report questionnaires and visualize both self-report and clinical evaluation questionnaires; 2) the physicians who can access their patients’ data and 3) the clinical and molecular curators who ensure the high quality of data stored in the database through a manual quality control. For each patient, multiple self-report and clinical evaluation questionnaires can be created, generally every eighteen months. Self-report questionnaires can only be filled by patients with FSHD1, while two versions of the clinical evaluation questionnaire are proposed, one for FSHD1 and one for FSHD2 patients.The legal approval for the French FSHD registry has been obtained from the CNIL  under authorization No. 912291 on June 25th, 2012. The CCTIRS  also gave a favorable opinion No. 12.004bis. Furthermore, a written informed consent was obtained from every patient before entering any data into the registry.Web access to the registry is secured by a two-factor authentication process combining a login and a password. An additional layer of security was added through the use of a session-specific token. Supplemental security measures have also been implemented, including login throttling and inactivity timeout.Daily backup and a log file are used to avoid loss of data in case of system failure.The French National Registry of patients with FSHD has been developed to collect clinical and molecular data obtained from two sources: a self-report questionnaire filled by the patient and a clinical evaluation questionnaire filled by both the patient and the physician providing follow-up care. The questionnaires are sent to the clinical curator who controls the data quality prior to their import into the database. Once the patient has been created, a unique Personal Identification Number (PIN) code is sent to the patient and to the physician if a clinical evaluation form was filled.As patients are followed within a highly structured national network of reference centers, physicians can be granted access to their patients’ data only or to patients registered in their reference center. In parallel, to simplify the patient’s mobility within the network of reference centers, each patient is affiliated to a specific group and can be moved from one center to another. Actors and their interplay are illustrated in Fig. Two levels of data curation are available: the clinical curator can create new patients and new self-reports or clinical evaluation questionnaires, while the molecular curator can only fill data in existing questionnaires. Both curators have access only to data related to their domain of expertise, i.e. clinics and genetics. A questionnaire is considered validated only when the two sections are validated. All mandatory items thus have to be filled in  and data quality (with an emphasis on missing data). The second set extracts gender- and age-related data. The third set displays the medical specialties of the physicians who include patients in the registry. The fourth set contains data related to the molecular diagnosis: number of patients per laboratory and diagnosis results. The last one provides a graphical display of the patients’ population based on selected clinical criteria.The FSHD French National Registry was approved in June 2012 and set up in 2013. Considering the FSHD prevalence  and the French population (65 millions), we can estimate that about 3000 FSHD patients are living in France. During the last 5 years, we collected clinical and molecular data from 638 FSHD patients over the country representing about 21% of the French FSHD population. Data from 26% of patients were collected only from self-report questionnaires; 14.9% from a clinical evaluation questionnaire; while for the remaining 59.1% of patients, data were collected from both questionnaires .The French FSHD registry includes various tools to visualize and analyze data. For instance, it is possible to display the number of concomitant affections among patients suffering or not from a given affection as illustrated in Fig. http://www.fshd.fr) containing the secured database itself with a freely accessible part. It contains general information on the French National Registry, the disease, the diagnosis, the associated French network centers, the state of research, reference publications and clinical trials. Links to other national registries are provided. Video and various documents are available and can be downloaded and used to join the registry. Newsletters also ensure regular information to patients about the registry evolution and advertise about forthcoming regional meetings with experts.Because this registry contains sensitive data, it was approved by the appropriate French review boards (CNIL and CCTIRS) and is hosted within a secured environment. The registry is accessible through a website  and the active participation of experts from the French national network for rare neuromuscular diseases (FILNEMUS), which is one of the 23 rare disease healthcare networks built through the second French national plan on rare diseases.To ensure interoperability at the global level, we use genetics standards coupled to clinical description. We therefore mapped these terms to ontologies, dictionaries and terminology systems to evaluate if one of the tested systems can be used for match-making . We obseThe French National Registry of FSHD patients is part of a national effort to develop national databases –25. ThesFSHD patients will benefit of the development and improvement of the French National FSHD Registry. Thanks to collected data, the registry will facilitate and accelerate clinical and translational research on the disease. Furthermore, specific guidelines on FSHD medical care will be regularly updated and disseminated, contributing to the national standardization of medical care for FSHD patients. Moreover, thanks to the registry, the assessment of feasibility and recruitment in clinical trials and studies will be facilitated. Despite strong efforts from physicians to ensure the collection of longitudinal data, this aspect is always a weakness in patients’ registries. In order to make it more efficient, we are currently modifying the system to allow the direct data submission from patients. The curators will then validate these new information to ensure their quality.The regular update of the website will contribute to the improvement of the knowledge of both patients and professionals on FSHD, as well as facilitate interaction with academic and industrial partners. In addition, through the website, users already have access to educational links and other relevant resources (for example FSHD meetings and congresses).Additional file 1:Figure S1. French National FSHD registry - SELF REPORT FORM (PDF 1194 kb)Additional file 2:Figure S2. French National FSHD registry - INCLUSION FORM (PDF 431 kb)Additional file 3:Figure S3. French National FSHD registry - INCLUSION FORM – FSHD LIKE (PDF 473 kb)Additional file 4:Table S1. Pre-filling rules for the self-report and the clinical evaluation questionnaires. (PDF 59 kb)Additional file 5:Table S2. Conflicts identification rules for the clinical evaluation questionnaire. (PDF 54 kb)Additional file 6:Figure S4. Network of reference centers contributing to the French FSHD registry (PNG 112 kb)Additional file 7:Table S3. Mapping of clinical terms to reference systems. CTO = the Clinical Trials Ontology; CTV3 = the Read Codes Clinical Terms Version 3; HPO = the Human Phenotype Ontology; ICECI = the International Classification of External Causes of Injuries; ICF = the International Classification of Functioning, Disability and Health; LOINC = the Logical Observation Identifier Names and Codes; MEDDRA = the Medical Dictionary for Regulatory Activities; MESH = the Medical Subject Headings; NCIt = the National Cancer Institute Thesaurus; NLMVS = the NIH NLM Value Sets; OMIM = the Online Mendelian Inheritance in Man; PHENX = the PhenX Phenotypic Terms; SMASH = the SMASH Ontology; SNOMED-CT = the Systematized Nomenclature of Medicine - Clinical Terms; XCO = the Experimental Conditions Ontology. (PDF 40 kb)


Reduction-oxidation (redox) reactions, in which electrons move from a donor to an acceptor, are the functional heart of photosynthesis. It is not surprising therefore that reactive oxygen species (ROS) are generated in abundance by photosynthesis, providing a plethora of redox signals as well as functioning as essential regulators of energy and metabolic fluxes. Chloroplasts are equipped with an elaborate and multifaceted protective network that allows photosynthesis to function with high productivity even in resource-limited natural environments. This includes numerous antioxidants with overlapping functions that provide enormous flexibility in redox control. ROS are an integral part of the repertoire of chloroplast signals that are transferred to the nucleus to convey essential information concerning redox pressure within the electron transport chain. Current evidence suggests that there is specificity in the gene-expression profiles triggered by the different ROS signals, so that singlet oxygen triggers programs related to over excitation of photosystem (PS) II while superoxide and hydrogen peroxide promote the expression of other suites of genes that may serve to alleviate electron pressure on the reducing side of PSI. Not all chloroplasts are equal in their signaling functions, with some sub-populations appearing to have better contacts/access to the nucleus than others to promote genetic and epigenetic responses. While the concept that light-induced increases in ROS result in damage to PSII and photoinhibition is embedded in the photosynthesis literature, there is little consensus concerning the extent to which such oxidative damage happens in nature. Slowly reversible decreases in photosynthetic capacity are not necessarily the result of light-induced damage to PSII reaction centers. The incorporation of the photosynthetic electron transport chain appears to have been accompanied by a large expansion of protein cysteine thiols used to sense and regulate cellular redox state . The opeablished . Moreoveablished .Light-driven redox changes in the photosynthetic electron transport system drive energy conversion that is harnessed in the forms of reducing power (reduced ferredoxin and NADPH) and ATP. The rate of carbon assimilation is matched to prevailing electron fluxes through the reduction of TRXs. Reduced TRXs then reduce disulphide bridges on target proteins within the Calvin cycle . ChloropThe ferredoxin-dependent TRX and NTRC systems function together with 2-cysteine peroxiredoxins (2-Cys PRX) as a two-component hydrogen peroxide-detoxifying system under stress conditions . 2-Cys PFor many years, the dominant concept in redox signaling has been that cells balance ROS on one side and antioxidants on the other . Accordi22O2 by the action of the thylakoid copper/zinc SODs. H2O2 can then be reduced to water by chloroplast APXs and the PRXs  and it is also reduced to H2O2 by ascorbic acid, which is abundant in chloroplasts. In the mitochondria, superoxide is able to inactivate the tricarboxylic acid cycle enzyme aconitase, which is a sensitive target in the matrix, but it has not been shown to cause inhibition of Calvin cycle enzymes that, in contrast, are very susceptible to oxidation and inactivation by H2O2. Hence, plastid H2O2 levels have to be very low and in the micromolar range. It is worthy of note that H2O2 and superoxide have divergent effects on cell survival and programmed cell suicide pathways in animals, where H2O2 promotes and superoxide inhibits cell death programs and apoptotic signaling .It is now well-established that ROS are key components of chloroplast‐nucleus retrograde signaling pathways, working together with a complex array of putative retrograde signaling molecules including metabolites and mobile transcription factors, calcium and links to pathways such as the unfolded protein response. Of these, the 3′-phosphoadenosine 5′-phosphate (PAP) phosphatase called SAL1 is perhaps the best characterized; SAL1 is thought to act as a sensor of oxidative stress in the chloroplasts because it undergoes a conformational change in response to redox changes. Chloroplast oxidation results in deactivation of SAL1 and accumulation of its substrate PAP, which acts as plastid signal instigating chloroplast-to-nucleus retrograde signaling pathways . It shouignaling . It may eristems . The sho2O2 production in systemic leaves that are distant from the apical leaves [?] leading to increased cyclic electron flow around PSI during photosynthetic induction , is a powerful oxidant that reacts rapidly with macromolecules in the vicinity of where it is generated, resulting in oxidation that is often referred to as “damage” (1O2 (de novo synthesis of D1 by targeting the protein elongation factor G (CpEF-G)  and other membrane lipids  . D1 is c(CpEF-G) . Howevere lipids . These pe lipids . Lipid pe lipids . Moreove and ch1 . However1O2 −triggered chloroplast-to-nucleus signaling pathways have been described to date: firstly, 1O2 may modify gene expression through interaction with β-carotene and the generation of carotenoid breakdown products (β-CC), some of which are volatile  and chlorina 1 (ch1) mutants, acting as key players in the decision between acclimation and cell death as they do in H2O2–mediated induction of programmed cell death observed in catalase (cat)2 mutants (flu mutants accumulate the chloroplast precursor protochlorophyllide (Pchlide) in the dark because conversion of Pchlide to Chlorophyllide is light-dependent. Pchlide molecules are potent photosensitizers that generate 1O2 in abundance upon illumination . Several processes that contribute to NPQ can be distinguished through their recovery kinetics. These are the state transition (qT), photoinhibition (qI), heat dissipation (qE) and zeaxanthin-dependent (qZ) quenching  and 1O2, resulting in a higher-energy triplet state (Car*), which can decay to the ground state via different reaction mechanisms such as intersystem crossing, triplet-triplet annihilation and ground state quenching  showed little increase in stress sensitivity compared to the wild type, crossing vte1 with the npq1 mutant that is deficient in violaxanthin deepoxidase, the enzyme responsible for the conversion of violaxanthin to zeaxanthin in the thylakoid membrane, resulted in oxidation of lipids and pigments, as well as photoinhibition . In its basic form, this pathway consists of a network of reactions involving APX and enzymes that thereafter serve to regenerate ascorbate. APX produces monodehydroascorbate radicals (MDHA), which can then be reduced back to ascorbate by ferredoxin or by NAD(P)H in a reaction catalysed by MDHA reductase  had impaired photosynthetic efficiency and showed signs of oxidation suggesting that the chloroplast 2-Cys PRXs are essential and perhaps more important than tAPX in preventing H2O2 accumulation. However, 2-Cys PRXs are also involved in the reduction of reactive lipid peroxides, thus providing additional protection to the chloroplasts. Interestingly, while the 2cpa 2cpb tapx triple mutants showed oxidative stress markers including expression of H2O2-responsive marker genes, leaf H2O2 levels were similar to those measured in the 2cpa 2cpb  and thylakoid (tAPX) forms of APX, however, revealed that the chloroplasts do not depended on the ascorbate-glutathione cycle alone to remove H2O2 . In contcpa 2cpb . These fvels low .vtc2-1 mutants, which have low ascorbate, showed higher levels of photoinhibition and photooxidation than the wild type under high light  was shown to transport ascorbate across the envelope membrane  and at lgh light . Howevergh light .Adaptation to biotic and abiotic threats in the environment is thought to have a long-lasting impact on plant processes that is passed to successive generations through persistent epigenetic mechanisms that provide a pre-emptive advantage. While a growing body of evidence suggests that changes in the reduction/oxidation (redox) status of stress signaling molecules and the level of DNA methylation play a major role in the transgenerational embedding of stress tolerance, little information is available concerning the specific pathways and mechanisms involved. The plant’s environmental sense and response mechanism contains a proteome distinct but overlapping with that of the photosynthetic chloroplast, with several stress proteins, including MSH1, appearing uniquely localized to the sensory plastid.5Chloroplasts are important sensors of environmental change, producing redox and other signals that can be transferred directly, or indirectly, to the nucleus to influence gene expression leading to acclimation to prevailing environmental conditions. Redox signals arising in chloroplasts also participate in epigenetic controls . Recent MSH1 pathway have been found to be localized in the epidermal and vascular parenchyma, as well as in reproductive cells. The sensory chloroplasts, which are about 30% of the size of photosynthetic chloroplasts and have decreased granal stacking, appear to be otherwise very similar to the more general chloroplast population, whose main function is photosynthesis, but they contain additional stress proteins, including MSH1, which associates with the thylakoid membrane and plastoglobuli (MSH1 produces a strong plant-stress response with differential expression of genes associated with abiotic and biotic stress pathways, together with genome-wide methylome re-patterning (msh1 memory is stable and heritable indefinitely.Adaptation to the environment has both short term and long-lasting impacts on plant processes. In some cases, such stress memories are passed to successive generations through persistent epigenetic mechanisms that provide a pre-emptive advantage in a changing climate. Crucially, cellular redox status is a regulator of the level of DNA methylation, which plays a major role in the transgenerational embedding of stress tolerance memories in animals and plants. Reprogramming of the methylome i.e. the set of nucleic acid methylation modifications in the genome, predisposes the next generation for enhanced stress responses. The redox state of the sensory chloroplast population may also play a pivotal role in the regulation of such epigenetic controls in plants. The specialized sensory chloroplasts that house the oglobuli . Silencitterning . Subsequtterning . The mshMSH1 is clearly associated with the redox functions of the chloroplasts. It is possible that the MSH1 protein is able to move from the chloroplast to the nucleus, as has been suggested for another plastid single-stranded DNA-binding protein, WHIRLY1 (2+ signalling and WHIRLY1 functions (The sensory function of  WHIRLY1 . The parunctions . It is lunctions .66f complex. This regulation extends as far as regulation of nuclear alternative splicing to adjust plant responses to varying light conditions (The driving force for photosynthesis, sunlight, is an almost limitless supply. Hence, energy conservation is not a key issue for photosynthetic regulation. A sophisticated network of mechanisms that dissipate energy are essential to the operation of light harvesting linked to electron transport in photosynthesis, allowing effective protection against excess irradiance. The flexibility of photosynthesis has become optimized during evolution allowing photosynthesis to operate over a wide range of irradiances and environmental variables. The photosynthetic processes have evolved to be highly plastic and flexible, allowing other processes such as ROS production to occur at the expense of efficiency. Multiple layers of overlapping protective systems serve to prevent damage to the photosynthetic machinery, via combined action of preemptive energy dissipation and ROS removal through antioxidants. Give the requirement to perceive fluctuations in factors that influence the effective operation of photosynthesis in order to regulate these protective mechanisms, it is not surprising that the chloroplast became a major sensor of environmental and metabolic change with the ability to transmit retrograde signals to the nucleus in order to make appropriate adjustments in gene expression. Direct regulation of plastome gene expression is achieved via activation of protein kinases through the reduction state of photosynthetic electron transport chain components, notably plastoquinone and the cytochrome bnditions . This innditions . While iDespite compelling evidence that ROS are pro-life signals with multi-faceted roles in plant growth and development, the concept that oxidative damage is a major cause of light-induced loss of cellular functions remains prominent in the photosynthesis literature. This may be in part due to the heavy reliance on measured decreases in Fv/Fm ratios as a measure of photoinhibition, without consideration that such changes may be related to sustained PSII downregulation via protective systems. Hence, caution should be exercised in the use of Fv/Fm ratios to measure of photo-damage .Chloroplasts are equipped with an abundance of antioxidants that clearly have overlapping functions as ROS targets. Some of these antioxidants, such as glutathione and PRX, have well characterized roles in signal transduction. While others, such as ascorbate, may simply serve to attenuate the ROS signal, the complexity of the chloroplast oxidant and antioxidant system affords an extensive repertoire of discrete and probably specific redox signals that provides enormous flexibility in the control of gene expression. This communication with the nucleus not only conveys essential information on redox pressure within the electron transport chain to triggers appropriate short-term genetic responses, but also allows strategic provision of future defenses through epigenetic modifications.


Marjolin's tumor is a term used to describe a malignancy developing in the setting of a chronic wound, infection, or other tissue subject to chronic inflammatory changes. These malignancies usually present after many years of chronicity, and can range from lower grade basal cell carcinomas to high-grade sarcomas. We present the case of a squamous cell carcinoma that developed within a chronic periprosthetic infection of a total knee arthroplasty of 7 years duration. The intra-articular location, association with an orthopaedic implant, and brief latency period are all unique features of this case. A 57 year-old female with a past medical history of rheumatoid arthritis on long-term hydroxychloroquine and leflunomide, as well as pulmonary embolism and hypothyroidism, underwent left total knee arthroplasty (TKA) on 9/16/2009. She developed an acute hematogenously-seeded periprosthetic joint infection (PJI) of unspecified organism and underwent debridement and exchange of polyethylene on 6/7/2011, followed by 6 weeks of parenteral antibiotics. She was asymptomatic after this procedure until 6/4/2012, at which time she developed a small cyst over the knee which was excised on 12/3/2012. This was complicated by wound healing difficulties, necessitating a split-thickness skin grafting procedure, which was then followed shortly by a second PJI. She underwent a 2-stage revision with explantation of components and antibiotic spacer insertion on 3/8/2013, followed by spacer removal and implantation of components on 6/4/2013. After persistence of PJI was suspected, she was referred to a musculoskeletal infection specialist for consultation  was 101 and c-reactive protein (CRP) was 87.2. Plain radiographs demonstrated progressive lytic changes of the tibia suggestive of abscess formation and osteomyelitis  is credited to Aurelius Cornelius Celsus in the first century, who described malignant changes in a burn scar.16Marjolin's ulcer has been classically associated with burns, reported to occur as frequently in 2% of chronic burn scars.3Any chronic wound that undergoes a change in appearance or drainage should alert providers to the possibility of malignant transformation. A comprehensive history detailing the chronicity of the wound as well as attempted treatments should be documented. Suspect lesions should undergo biopsy performed by or at the direction of the treating surgeon.10Once the diagnosis of Marjolin's ulcer is confirmed, collaboration with a multidisciplinary oncology team will ensure that general recommendations proposed by the AJCC will be followed.Our case report presents several unique features to the Marjolin's tumor. This mass developed as a consequence of long-standing periprosthetic joint infection in an immunosuppressed host, with a latency period of only 7 years, and an even shorter period of off-and-on wound drainage. Furthermore this malignancy developed in an intraosseous and intra-articular location, without superficial skin involvement. The case patient was fortunate, in that her locoregional lymphadenopathy was reactive in nature secondary to infection, and not consistent with metastatic spread. Ablation of the extremity was therefore potentially curative.Marjolin's Ulcers are frequently aggressive tumors complicating chronically infected wounds and tissues of the extremities. To our knowledge, the incidence of malignant transformation in chronic periprosthetic joint infection is unknown. This unusual complication was identified intraoperatively by the recognition of a tissue bed with an atypical appearance, which highlights the importance of maintaining a high index of suspicion when managing chronic musculoskeletal infection. Consistent with the adage “culture what you biopsy and biopsy what you culture,” this case confirms that it is prudent to send material for histopathologic examination when performing debridement procedures, particularly when the appearance of the wound bed or drainage have changed.


Lynch syndrome is a hereditary cancer syndrome caused by constitutional pathogenic variants in the DNA mismatch repair (MMR) system, leading to increased risk of colorectal, endometrial and other cancers. The study aimed to identify the incremental costs and consequences of strategies to identify Lynch syndrome in women with endometrial cancer.A decision-analytic model was developed to evaluate the relative cost-effectiveness of reflex testing strategies for identifying Lynch syndrome in women with endometrial cancer taking the NHS perspective and a lifetime horizon. Model input parameters were sourced from various published sources. Consequences were measured using quality-adjusted life years (QALYs). A cost-effectiveness threshold of £20 000/QALY was used.MLH1 methylation testing was cost-effective versus no testing, costing £14 200 per QALY gained. There was uncertainty due to parameter imprecision, with an estimated 42% chance this strategy is not cost-effective compared with no testing. Age had a significant impact on cost-effectiveness, with testing not predicted to be cost-effective in patients aged 65 years and over.Reflex testing for Lynch syndrome using MMR immunohistochemistry and MLH1 methylation testing may be cost-effective. Age cut-offs may be controversial and adversely affect implementation.Testing for Lynch syndrome in younger women with endometrial cancer using MMR immunohistochemistry and  Lynch syndrome (LS) is a hereditary cancer syndrome caused by pathogenic variants (mutations) in the mismatch repair (MMR) genes which leads to an increased risk of cancers, particularly colorectal cancer (CRC) and endometrial cancer (EC) . Around reflex testing. When a pathogenic variant is identified, predictive testing can be offered to relatives, usually through cascade testing to minimise the number of wasted tests.LS is typically identified through cancer-affected individuals and testing is typically driven by tumour-based triage tests. The automatic application of these tests to eligible patients is termed Interventions to reduce the risk of cancer incidence and mortality for families with LS include surveillance, risk-reducing surgery, and aspirin chemoprevention .BRAF V600E, useful in CRC, has no utility in EC.Reflex testing for LS is cost-effective in CRC –6 and isWe know of three economic evaluations of reflex testing for LS in women with EC, none in the NHS setting, and none including a no testing comparator –10.The aim of this study is to identify the relative cost-effectiveness of reflex testing for LS in women with EC in the NHS.Table 1and is reported in line with published criteria [This study used methods in line with the NICE reference case  to addrecriteria .Reflex testing is conducted in women newly diagnosed with EC (probands), so the population includes all women diagnosed with EC even if they do not have LS. Relatives may also subsequently be diagnosed with LS by predictive testing, so all relatives who could feasibly be diagnosed are modelled in the population, even if neither they nor the proband have LS or receive testing (maintaining a consistent population across interventions).The prevalence of LS among probands declines with age, as do the opportunities to intervene meaningfully, therefore cost-effectiveness is sensitive to the age of probands. The model simulates a cohort of probands of a specific age (60 years in the base case) so that the impact of age on cost-effectiveness can be quantified.The age distribution of relatives also affects cost-effectiveness, since younger relatives have a greater opportunity to benefit from interventions. In the base case analysis and scenario analyses we use a weighted average of the results considering the full distribution of relatives’ ages, while in the probabilistic and one-way sensitivity analyses relatives start the model aged 54 as this produces cost-effectiveness results closest to a weighted average of results across relatives’ ages.Five reflex testing strategies were identified following consultation with clinical experts, plus a no testing option (representing current practice). Four strategies utilised tumour tests to triage; the fifth involved direct referral to genetic counselling for mutation testing. We did not include the use of tools to predict the risk of LS  as interWe modelled the following care pathways:Diagnostic testing in women with EC to identify those with LS;Genetic testing of male and female relatives of those women with LS to identify further cases of LS;Biennial colonoscopic surveillance for CRC for those having, or suspected of having LS.The model focussed on CRC outcomes for patients as this has a significantly greater burden in terms of mortality for people with LS than other cancers. For comparison, in a prospective study of LS, incidence and 5-year survival figures suggest 4.5 deaths from colorectal cancer for every 1 death from ovarian cancer  , putative LS  and sporadic. Women diagnosed with (P)LS were offered interventions to reduce the incidence and mortality of CRC. Relatives of women diagnosed with LS were offered predictive mutation testing, with risk-reducing interventions offered to those with the variant. First-degree relatives of women diagnosed as PLS were invited to genetic counselling and offered biennial colonoscopic surveillance.Fig 1) where the long-term outcomes (costs and QALYs) were estimated using a Markov model .The model was a decision tree .Clinical data were sourced from published literature through pragmatic literature review . When studies at risk of bias due to high dropout (≥ 10%) were excluded, the estimated prevalence was reduced to 3.0%.The prevalence of LS in EC was estimated based on results from sixteen studies of testing for LS in endometrial cancer , 17–31 aMLH1 and MSH2 are associated with earlier age of onset of endometrial cancer than mutations in MSH6 and PMS2 and non-Lynch syndrome endometrial cancer. These mutations may also not be evenly distributed in the population. To estimate the distribution of gene mutations for probands of a given age, we estimated the overall relative abundance of the genes in endometrial cancer patients (of any age) by pooling studies reporting gene distributions and imposing an age limit for testing of 70 years or higher or no limit. Sixty-five endometrial cancer patients with identified MMR mutations across eight studies [MLH1—MSH2—MSH6—PMS2. The age-specific relative abundance was then estimated by considering the age-dependent risks of endometrial cancer reported from the Prospective Lynch Syndrome Database (PLSD) [Table 2.Lynch syndrome gene mutations in  studies , 28, 31 e (PLSD) . A restrProbands enter the model with EC and the associated mortality risk. Survival of EC is better for women with LS compared to those with sporadic EC. The mortality rate for women with LS was estimated as 4.0 per 1000 person years based on an analysis from prospectively registered women with LS . The morMLH1 mutations were predicted to have the highest risk, with MSH2, MSH6 and PMS2 mutation carriers having progressively lower risks (S3 Appendix).To estimate the incidence of CRC in individuals with LS, survival models were fitted to reported data from the Prospective Lynch Syndrome Database (PLSD) , 39, 40.These estimates were used for individuals with LS undergoing surveillance; CRC incidence for individuals with LS not receiving surveillance was estimated by applying the (inverted) hazard ratio for incidence due to surveillance colonoscopy (see below) to these estimates.CRC incidence for individuals without LS was estimated from national statistics .CRC mortality for individuals without LS was estimated from national 5-year survival statistics .CRC survival is improved for individuals with LS, except for metastatic disease. The CRC mortality rate for individuals with LS was estimated by applying a hazard ratio of 0.66 for Stage I–III CRC .For each tumour-based test, the sensitivity, specificity and test failure rate were estimated.S4 Appendix for details of methods and results).Meta-analyses of sensitivity and specificity were performed using the bivariate method  without Eight studies , 25, 30 MLH1 mutation [MLH1 mutations [The proportion of positive IHC results with absent/abnormal staining of MLH1 was assumed to be 89% in those with an mutation , 31, 44,utations , 31, 44,utations , 31, 44.Eight studies , 25, 30 The sensitivity and specificity of MSI were estimated as 90.3% and 77.1%. The test failure rate was estimated from six studies , 24, 25 MLH1 methylation testing following abnormal IHC results for MLH1. As no studies discovered methylated tumours with germline mutations, it is not possible to estimate the sensitivity through meta-analysis. A number of studies did not test for germline mutations if MLH1 methylation was identified in the tumour, but two did [MLH1 mutations do not show methylation. Specificity was estimated as 93.6%, applying to those without MMR mutations and those with MSH2, MSH6 and PMS2 mutations.Seven studies , 28, 31  two did , 25, and two did  blood DNMLH1 methylation testing following detection of MSI [MLH1 methylation testing also performed IHC and only performed MLH1 methylation testing if MLH1 staining was absent/abnormal. Therefore, we assumed for patients with an MLH1 mutation the sensitivity would be equivalent in patients with MSI as those with dMLH1 (95%). For patients with mutations in MSH2, MSH6 and PMS2, we estimated that 1/14 would demonstrate MLH1 methylation and be inappropriately discharged from further testing [MLH1 methylation and be discharged from further testing [Only one study estimated the performance of n of MSI , since a testing . For pat testing . In a sc testing  for thesMLH1 methylation). Most of these LS-causing mutations are readily detected, although some may be more challenging to identify. Interpretation is coordinated internationally [Mutations causing LS are numerous and heterogeneous. Furthermore, heritable mutations can have epigenetic effects on the MMR genes . It is further assumed the sensitivity of diagnostic mutation testing is 90%, i.e., 10% of LS-causing mutations are not identified.It was assumed predictive mutation testing is 100% accurate.It was assumed 55% of EC patients with tumour-based test results suggestive of LS would attend genetic counselling , and 10%The effectiveness of colonoscopic surveillance is a key determinant of the cost-effectiveness of strategies to identify LS , 5.Ladabaum et al.  reviewedMLH1 cases is more effective [Estimates from a study by Järvinen et al.  have beeffective . Of the ffective , the stuffective  estimatiEstimates for the impact of colonoscopic surveillance on CRC incidence were therefore estimated from Järvinen et al. , 51 in tCRCs were assumed to be detected in earlier stages in those undergoing surveillance .Consistent with the NICE reference case , we sougBaseline utility values (according to age and sex) were estimated from population norms , with imThere is little evidence that non-metastatic CRC is associated with a lower  utility than population norms . TherefoAlthough health-related quality of life may be significantly worsened in a small minority of EC patients, it was assumed that on average there would be no disutility from population norms.Genetic testing can have a number of different effects on what would broadly be considered utility, although its impact on health-related quality of life is more commonly investigated in terms of the impact on anxiety, depression and mood , 62.A pragmatic search of Embase and MEDLINE using a search filter for utility values  failed tIn the base case analysis, it was assumed that there would be no direct impact on QALYs from genetic counselling or genetic testing.Costs are presented in 2016/17 pounds Sterling , inflated using the Hospital and Community Health Services Pay and Prices Index  to 2015/IHC, MSI and methylation were estimated to cost £210, £202 and £136  as averaThe cost of offering counselling to a proband was estimated as £27 . The cost of referral for a relative was estimated as £36  .Pre-test genetic counselling was estimated to cost £347 and £172 for probands and relatives respectively, and post-test genetic counselling was estimated to cost £133 .The cost of diagnostic mutation testing for LS was estimated as £755 . The cost of predictive mutation testing was estimated as £166 , with no further cost being accrued due to time in CRC states or at time of death from CRC.The source of cost estimates is a report by the Economic Evaluation of Health and Social Care Interventions Policy Research Unit , based oS5 Appendix provides a full listing of model input parameters.Fully incremental analyses are performed throughout.A base case deterministic analysis was conducted, holding all model input parameter values fixed at a central estimate. A probabilistic sensitivity analysis (PSA) was conducted, in which parameter values were simultaneously varied according to distributions reflecting their uncertainty . One-wayAll testing strategies were predicted to result in net QALY gains and increased costs versus no testing.MSI with methylation was predicted to result in the least QALY gains of the testing strategies, due to diagnosing the lowest number of people with LS or as PLS. IHC-based strategies were predicted to result in greater QALY gains than equivalent MSI-based strategies as they were more sensitive. Methylation-based strategies were predicted to result in lower QALY gains than equivalent strategies without methylation testing as they are less sensitive.IHC with methylation was the testing strategy predicted to have the lowest total cost. IHC without methylation was predicted to have the highest total cost. The use of methylation testing substantially reduced costs.Table 3).Only IHC with methylation would be considered cost-effective at a threshold of £20 000 per QALY (The age of probands was varied from 40 to 85 years (not changing the distribution of the ages of relatives). The economic values of all testing strategies strictly decreased with increasing proband age. Across this range, IHC with methylation produced the most net health benefit of the testing strategies (at a willingness to pay of £20 000 per QALY), although the incremental net health benefit of this strategy (versus no testing) became negative for probands aged 65 years and upwards.To examine the policy impact of heterogeneity, we estimated the cost-effectiveness of testing with IHC and methylation versus no testing when the population is individuals aged up to a particular age threshold. This analysis suggests that maximum economic value is achieved when the age threshold is somewhere between 60 and 65 years, but that using an age threshold of 70 years would produce more economic value than a threshold of 50 years, and that even with an age threshold of 80 years there is still positive economic value versus no testing. This happens because testing in younger endometrial cancer patients subsidises the testing in older patients.Table 2). There is decision uncertainty due to parameter imprecision, since IHC with methylation is only the optimal strategy in 36% of iterations at a threshold of £20 000 per QALY . The 95% credible interval for the incremental net monetary benefit of IHC with methylation versus no testing is −£436 to £2204 per proband, and it was negative in 42% of iterations.A probabilistic sensitivity analysis (PSA) was conducted with 1000 iterations. The mean results were consistent with the base case analysis, in that only IHC with methylation was predicted to be cost-effective at a threshold of £20 000 per QALY (Another PSA was conducted with the age of probands set to 50 (instead of the base case 60 years). IHC with methylation was predicted to be the optimal strategy in 45% of iterations, and to be cost-effective compared to no testing in 90% of iterations.Fig 4). The most influential parameters are the age of the proband and the effectiveness of colonoscopy in reducing CRC incidence. Six parameters had the potential to make testing not cost-effective within the ranges considered.One-way sensitivity analyses were conducted with lower and upper parameter values equal to the 95% confidence limits for each parameter in the probabilistic sensitivity analysis . When risk prediction tools were included as testing options, these were all less effective (produced fewer QALYs) than the existing testing strategies, and they were all dominated or extendedly dominated and so would not be considered cost-effective.A number of scenario analyses were conducted to examine key parameter and structural assumptions. In all but one of these scenario analyses, IHC with al. 2005 , none ofMLH1 methylation testing. Stronger conclusions cannot be drawn from the current analysis as there remains a substantial possibility that testing  is less cost-effective than no testing. At a lower age limit (50 years), there can be reasonable confidence that testing with IHC (or MSI) with methylation testing is cost-effective compared to no testing. Of interest, however, is the finding that the optimal programme (from a cost-effectiveness perspective) would test all endometrial cancer patients up to an age threshold somewhere between 60 and 65. Furthermore, programmes testing all endometrial cancer patients up to age 70 or 80 years would also be cost-effective compared to no testing, even though they would not be cost-effective compared to a programme using an optimised age threshold.This economic evaluation found that testing for LS in EC patients may be cost-effective in women aged 60 using MMR immunohistochemistry and MLH1 methylation testing would be cost-effective compared to testing EC <50 years or using the revised Bethesda guidelines. Kwon et al. [Three studies have previously addressed the cost-effectiveness of testing for LS in EC. Goverde et al.  conducten et al.  used a Mn et al.  used a dOur study adds significantly to the literature: it provides an estimate of cost-effectiveness in the UK setting, it includes a no testing option (important given that current practice in most settings is no testing) and measures health effects in QALYs, as opposed to life years gained or cases detected.Our study has limitations; only CRC has been included as a downstream cancer, and we have not included risk-reducing measures besides colonoscopy. Aspirin is cheap and effective at lowering the risk of LS-associated cancers  and propOur study also assumes that there is no testing for MMR deficiency in endometrial cancer in the no testing comparator. It may be that this testing is already being done, or will be done in the near future, to select patients for targeted immune therapies . In thisIHC is conducted to a high standard in the UK, with use of external quality assurance. It is likely that published studies based in research centres will have similarly high standards, but routine clinical settings outside the UK may have lower standards. We have also assumed (based on the literature) that IHC would use all four MMR proteins implicated in LS, although it may be argued that testing for MSH6 and PMS2 expression could be cheaper and equally effective. If a 2-protein IHC panel were equally effective and 35% cheaper than a 4-protein panel, the ICER would reduce to £12,100 per QALY compared to no testing.We have not included any genetic testing for somatic MMR mutations, which is sometimes used  to confirm that a MMR deficient tumour with no constitutional pathogenic variant identified has arisen due to somatic MMR mutations rather than from Lynch syndrome. This would be expected to increase costs. An alternative strategy for testing could include testing for pathogenic MMR variants in tumour and normal colon tissue and only referring to clinical genetics when the results suggest a constitutional pathogenic variant (which would then be confirmed by further testing). If this testing could be performed for substantially less than the cost of genetic counselling it could be cost-effective, but current estimates do not support this being the case.There is a need for high-quality estimates of the effectiveness of surveillance colonoscopies. Although the use of randomised controlled trials is unsuitable, this does not preclude other study designs. As colonoscopies have cost implications for health services, often lead to discomfort and occasionally serious complications for those receiving them, it is important their true value in this setting is ascertained. We recommend research into the uptake of genetic counselling and testing in the UK in the context of reflex tumour-based testing. The current study assumes nearly half of patients decline genetic counselling, but this is based on a study in an insurance-based healthcare system, and could lead to an underestimate of the cost-effectiveness of testing. As shown in the one-way sensitivity analysis, if only 9% decline counselling following tumour testing, the incremental net benefit of testing is more than doubled .We recommend that concerns of cost-effectiveness should not be a barrier to implementing reflex testing for LS in young women with EC. We recommend that the use of an age limit is justified as older women (and their relatives) have less potential to benefit from testing, as the likelihood of a positive test result is lower. Where the use of age limit is unacceptable, further research should be conducted into the cost-effectiveness of testing before it is performed in all EC patients.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S4 Appendix(DOCX)Click here for additional data file.S5 Appendix(DOCX)Click here for additional data file.S6 Appendix(DOCX)Click here for additional data file.S7 Appendix(DOCX)Click here for additional data file.