Alzheimer’s disease (AD) is the most common age-related dementia. Besides its typical presentation with amnestic syndrome at onset, atypical AD cases are being increasingly recognized, often in presenile age.To provide an extensive clinical and genetic characterization of six AD patients carrying one or more singular features, including age of onset, atypical phenotype and disease progression rate. By reviewing the pertinent literature and accessing publicly available databases, we aimed to assess the frequency and the significance of the identified genetic variants.Biomarkers of amyloid-β deposition and neurodegeneration were used to establish the in vivo diagnosis of probable AD, in addition to neurological and neuropsychological evaluation, extensive laboratory assays and neuroradiological data. Considering the presenile onset of the majority of the cases, we hypothesized genetically determined AD and performed extensive genetic analyses by both Sanger sequencing and next generation sequencing (NGS).PSEN1 and the other in PSEN2, and a novel silent variant in PSEN2. Most notably, we identified several additional variants in other dementia-related genes by NGS. Some of them have never been reported in any control or disease databases, representing variants unique to our cases.We disclosed two known missense variants, one in This work underlines the difficulties in reaching a confident in vivo diagnosis in cases of atypical dementia. Moreover, a wider genetic analysis by NGS approach may prove to be useful in specific cases, especially when the study of the so-far known AD causative genes produces negative or conflicting results. From a clinical perspective, AD typically displays an amnestic syndrome of the hippocampal type that can be associated with various cognitive or behavioural deficits during disease evolution . AtypicaAMY-PET) . ApproxiPSEN1 and the other in PSEN2, and a novel silent variant in PSEN2. Moreover, additional variants in dementia-related genes have been identified by next generation sequencing (NGS). Our results are intriguing as they raise the question of the role of genetic risk burden in AD.Here, we describe a case series of cognitive disorders with in vivo biomarker positivity for Aβ deposition, showing various clinical atypical aspects together with peculiar genetic features. We disclosed two known missense variants, one in 18FDG-PET) neuroimaging. The research for AD pathophysiological biomarkers, either CSF Aβ42, T-tau and P-tau assay or amyloid tracer PET, was performed in all patients.We describe 6 unrelated cases affected by cognitive disorders who underwent a complete diagnostic protocol including neurological and neuropsychological evaluation, extensive laboratory assays, EEG, structural (CT or MR) and functional  and in the following months psychomotor slowing. Familial history was negative. Neurological examination disclosed an asymmetric parkinsonian syndrome (R > L). MMSE was 22/30. Neuropsychological testing revealed deficits in verbal memory, attention, abstract reasoning and semantics. Brain MRI showed moderate atrophy in frontal, lateral temporal and temporo-mesial cortex prevailing on the left side. AMY-PET evidenced a massive and diffuse burden of amyloid-β. His extrapyramidal syndrome showed satisfying response to L-Dopa administration.18FDG-PET  [42 ratio was 7.533 (n.v. ≤ 0.52), whereas P-tau/Aβ42 was 1.031 (n.v. ≤ 0.08) [This subject developed at age 45 a complex behavioural syndrome characterized by apathy, social withdrawal, and eating and sleep disorders. Familial history was negative. Over the next 5 years, there was a clinical worsening with word-finding difficulties, dyscalculia, memory deficits and motor clumsiness in both hands. At age 50, his MMSE score was 16/30 and he presented ideo-motor apraxia, anomia, verbal memory deficits and dysexecutive syndrome. EEG showed diffuse slowing of cerebral electric activity, while brain MRI detected atrophy in parietal regions with slight right prevalence. Subsequently, he also manifested limb myoclonus and psychomotor agitation with complex visual hallucinations. Seven years after the symptom onset, the patient came to our observation and underwent a more extensive diagnostic protocol. Neurological examination showed left pyramidal and bilateral asymmetric (L > R) extrapyramidal syndrome, action-induced limb myoclonus and Epstein sign. MMSE score was 8/30. Brain MRI evidenced marked and diffuse atrophy, with posterior predominance. 0 pg/mL) . T-tau/A18FDG-PET , whereas T-tau and P-tau were normal; T-tau/Aβ42 and P-tau/Aβ42 ratios were 1.247 and 0.145 respectively. The research of 14.3.3 protein in CSF was negative.This case, without family history for cognitive disorders, insidiously presented at age 52 with a language disorder characterized by anomia and apraxia of speech which progressively worsened until mutism. Neurological examination showed a “worried” facial expression, asymmetric (R > L) mixed pyramidal and extrapyramidal syndrome, focal and segmental myoclonus, exaggerated startle reaction and frontal release signs. A neuropsychological examination showed severe non-fluent aphasia with almost complete mutism and slightly impaired comprehension, severe bucco-lingual and ideo-motor apraxia. EEG showed marked slowing in cerebral electric activity. Brain MRI revealed asymmetrical cortico-subcortical atrophy prevailing in left fronto-temporal areas. Written informed consent was acquired from all patients for genetic analysis, processing data and permission to publish data in respect of privacy.42 were determined with human specific ELISA kits (Innogenetics). Plasma level of progranulin was measured using an ELISA kit .CSF levels of T-tau, P-tau and AβAPP, PSEN1 and PSEN2 genes [APOE genotyping [APP, PSEN1, PSEN2, PRNP, GRN, MAPT, CHMP2B, FUS, TARDBP, VCP, TREM2, ABCA7, APOE, BIN1, CALHM1, CCL2, CCNF, CD33, CHCHD10, CLU, CSF1R, CST3, CTSF, DCTN1, FLNC, hnRNPA1, hnRNPA2B1, ITM2B, LRRK2, NCSTN, NOS3, NOTCH3, OPTN, PFN1, PLD3, PRKAR1B, SERPINI1, SIGMAR1, SNCA, SNCB, SORL1, SQSTM1, STH, TBK1, TMEM106B, TUBA4A, TYROBP, UBQLN2. Sequencing was performed on the Illumina MiSeq instrument using 2X150 bp paired-end read cycles. MiSeq Reporter software (Illumina) was used for alignment (reference human genome UCSC hg19) and variant calling. Variants were annotated using Variant Studio software (Illumina). Low-quality variants were filtered out using the Illumina Qscore threshold of 30; in addition, variants with a minor allele frequency higher than 2% in GnomAD  were filtered out. Variants of interest were confirmed using standard Sanger sequencing.Sanger Sequencing of N2 genes , 9 and Anotyping  was perfhttps://cadd.gs.washington.edu/) was used to predict the pathogenicity of a truncating variant (SORL1 Ser10STOP). NetGene2 (http://www.cbs.dtu.dk/services/NetGene2/) and BDGP (http://www.fruitfly.org/seq_tools/splice.html) splice site prediction tools were used to predict the effect on the splice site of the DCTN1 c.3529 + 5G > A variant.Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen) softwares were used to predict pathogenicity of missense mutations. Combined annotation-dependent depletion (CADD) score . Given the peculiarity of disease onset, plasma progranulin dosage was performed in cases 4, 5 and 6, with normal values. In addition, all patients underwent an APOE genotyping, which only in cases 2 and 3 showed a ε3/ε4 heterozygosity. The diagnosis of probable AD was supported by at least one positive pathophysiological biomarker in all cases: AMY-PET in cases 1, 2, 3 and 4 and CSF biomarkers in cases 5 and 6. In case 6, although T-tau and P-tau values were not increased, T-tau/Aβ42 and P-tau/Aβ42 ratios both resulted well above the standardized cut-offs [Our series consists of six cases whose clinical features are summarized in Table APP, PSEN1, PSEN2, PRNP, GRN, MAPT, CHMP2B, FUS, TARDBP, VCP, TREM2) were sequenced at 100% by NGS (read depth ≥ 20X) or, in some cases with incomplete coverage, by standard Sanger technique. Genetic results are described in Table PSEN1 (patients 1 and 2) and Arg71Trp in PSEN2 (patient 3), and a novel silent variant, Ser236Ser in PSEN2 (patient 4). Moreover, thanks to NGS approach, we disclosed other variants in dementia-related genes, in particular FUS, ABCA7, CSF1R, DCTN1, SERPINI1 and SORL1  or disease (HGMD) databases, representing variants unique to our cases. CADD analysis of the Alzheimer’s disease is mainly distinguished in a typical presentation with hippocampal amnestic syndrome and atypical forms with different cognitive or behavioural deficits.atypical” features including age at onset, clinical presentation and disease progression rate. Case 5 presented a complex syndrome indicative of bvFTD with parkinsonism and additional atypical features. The severity of clinical picture and the high levels of CSF tau might suggest the possibility of a prion disease. However, the long course, the MRI features, the neuroimaging findings  and CSF Aβ42 reduction made presenile AD the most likely diagnosis. Case 6 was classified as possible CBS, a clinical syndrome with different underlying pathological substrates [18FDG-PET hypometabolic pattern suggested an underlying AD pathology (CBS-AD), in agreement with the results of a recent combined 18FDG-PET/neuropathological study [18FDG-PET, MRI), in atypical dementia cases.In this paper, we describe a series of 6 unrelated patients affected by dementing syndromes characterized by one or more “bstrates , 12. In al study . NotablyPSEN1 Glu318Gly, was first identified in patients with EOAD [PSEN2 Arg71Trp, probably involved in protein stability and signalling pathways [PSEN1 Glu318Gly and PSEN2 Arg71Trp increase disease risk and modulate clinical phenotype. PSEN2 Ser236Ser, present in case 4, is a silent variant whose pathogenicity is not predictable.The results of genetic analyses were, in our opinion, very interesting. The variant found in cases 1 and 2, ith EOAD . Studiesith EOAD , 16, andith EOAD . The varpathways , 20, andpathways , 22. It ABCA7 and SORL1 are well-known AD risk genes [ABCA7 Asp679Tyr and the SORL1 Thr833Ile variants. They had never been reported before but are predicted to be deleterious by in silico analyses, therefore possibly exerting a synergistic effect with the PSEN1 Glu318Gly variant in amyloidogenic process.Among the relevant findings of NGS analysis, sk genes , 24. Thesk genes . A stronsk genes . A reducsk genes , has beesk genes . In patiFUS, a gene implicated in ALS and FTD cases [Patient 2, affected by LOAD with parkinsonism, harboured the Tyr239Cys variant in TD cases . This vaDCTN1 gene. Several DCTN1 mutations have been described in association with ALS, degenerative parkinsonisms and Perry syndrome [DCTN1 Iso196Val variant has been reported both in patients and in several healthy controls, making it a possible risk factor rather than a causative mutation. This patient also presented the Ser10STOP variant in SORL1, which is a truncating variant absent in ExAc  and GnomAD databases, with a CADD score of 35: these types of variant are considered as definitely pathogenic and associated with a significant 12-fold increased AD risk, which is comparable with the APOE-ε4 homozygosity effect [SORL1 mutations segregate with disease in LOAD families, and their pathological mechanism is likely to be haploinsufficiency [In patient 5, we found the Iso196Val variant in syndrome , 31. Intsyndrome , DCTN1 Iy effect . Rare paficiency .CSF1R. CSF1R mutations are causative of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia [CSF1R may influence the susceptibility to AD, as already shown for other adult-onset leukodystrophy causative genes, such as TREM2 and NOTCH3 [SERPINI1 are responsible for familial encephalopathy with neuroserpin inclusion bodies [SERPINI1 mutations [DCTN1 gene is predicted as potentially capable of altering the splicing site by in silico analyses; therefore, a possible pathogenic effect cannot be excluded.In case 6, we found variants in other dementia-related genes. The novel Ala273Thr variant, predicted as damaging by in silico analysis, was identified in ted glia , and havted glia . Noteword NOTCH3 , 38. Mutn bodies . Though utations , the Alaatypical” clinical aspects, despite the application of very extensive diagnostic protocols. Therefore, post-mortem neuropathological examination remains the gold standard to definitely elucidate the nature of the neurodegenerative process in the single patient with atypical dementia.In conclusion, two relevant aspects emerge from the observations made on this case series. First, some of the patients here presented are paradigmatic of the difficulties in reaching a confident in vivo diagnosis due to the “Second, in this series of cases, it is also possible to highlight the very interesting aspects emerging from a wider than standard genetic analysis. We found the coexistence of more than one rare non-causative genetic variant in 4 out of 6 patients, suggesting an additive contribution of them to develop dementia, whereas each single variant may not be sufficient. This raises a crucial question: what is the role of these non-causative mutations that are increasingly found in different neurological disorders, particularly in dementias? One hypothesis is that they could act as risk or modifier factors to the disease. Further studies adding evidence from NGS data to the current knowledge will be necessary to support this hypothesis and to define the individual risk associated to each variant.


The maximum values (%) of oleic acid (C18:1), palmitic acid (C16:0), UFAs, and the UFA/saturated fatty acid (SFA) ratio were found in bee pollen harvested during summer. Bee pollen harvested during spring ranked second in its oleic, palmitic, linolenic, stearic, arachidic, behenic, and lignoceric acid concentrations and for EFAs, UFAs, and the UFA/SFA ratio. It was concluded that the FA composition of bee pollen varied among the harvest seasons. We recommend harvesting pollen loads during spring and summer to feed honeybee colonies during periods of scarcity and for use as a healthy, nutritious food for humans.Harvesting pollen loads collected from a specific botanical origin is a complicated process that takes time and effort. Therefore, we aimed to determine the optimal season for harvesting pollen loads rich in essential fatty acids (EFAs) and unsaturated fatty acids (UFAs) from the Al-Ahsa Oasis in eastern Saudi Arabia. Pollen loads were collected throughout one year, and the tested samples were selected during the top collecting period in each season. Lipids and fatty acid composition were determined. The highest values of lipids concentration, linolenic acid (C18:3), stearic acid (C18:0), linoleic acid (C18:2), arachidic acid (C20:0), the sum of the C18:0, C18:1, C18:2, and C18:3 concentrations, and EFAs were obtained from bee pollen harvested during autumn. The maximum values (%) of oleic acid (C18:1), palmitic acid (C16:0), UFAs, and the UFA/saturated fatty acid (SFA) ratio were found in bee pollen harvested during summer. The highest concentrations (%) of behenic acid (C22:0), lignoceric acid (C24:0), and SFAs were found in bee pollen harvested during winter. Bee pollen harvested during spring ranked second in its oleic, palmitic, linolenic, stearic, arachidic, behenic, and lignoceric acid concentrations and for EFAs, UFAs, and the UFA/SFA ratio. The lowest SFA concentration was found in bee pollen harvested during summer. Oleic, palmitic, and linolenic acids were the most predominant FAs found in bee pollen. It was concluded that the FA composition of bee pollen varied among the harvest seasons due to the influence of the dominant botanical origins. We recommend harvesting pollen loads during spring and summer to feed honeybee colonies during periods of scarcity and for use as a healthy, nutritious food for humans.Seasonal variations in the fatty acid (FA) compositions of pollen loads collected from the Al-Ahsa Oasis in eastern Saudi Arabia throughout one year were determined to identify the optimal season for harvesting bee pollen rich in essential fatty acids (EFAs) and unsaturated fatty acids (UFAs). The highest values (%) of lipids, linolenic acid (C Pollen is the primary source of proteins, lipids, macro-and trace elements, vitamins, and other micro-components that are required for the development of honeybee colonies ,2,3,4,5.Paenibacillus larvae larvae) and European foulbrood (Melissococcus pluton) pathogens, as well as other microbes that may cause foulbrood [The lipid contents in dried bee pollen vary and range from 1.82% to 5.47% ,21,22,23oulbrood .Long-chain n-6 and n-3 polyunsaturated fatty acids (PUFAs) are synthesized from linoleic acid and alpha-linolenic acid, respectively . An EFA Pollen collection varies widely among different seasons and is affected by the availability of major pollen sources ,37,38,39Apis mellifera carnica) colonies (each of 10 combs) at five distinct apiaries  [The differences among the seasons were examined by one-way analysis of variance ANOVA), which indicated significant differences among the seasons. The normality in data was examined by the Shapiro–Wilk normality test, which indicated the normal distribution of the data. Therefore, the analysis was performed on the original data. The ANOVA was used to assess differences among the seasons, and correlations between fatty acids the SASNC, USA) . The treNC, USA) .Brassica napus L.), summer squash (Cucurbita pepo Thunb), date palm (Phoenix dactylifera L.), sunflower (Helianthus annuus L.), and alfalfa (Medicago sativa L.).The data presented in p < 0.01) varied depending on the harvest season. The concentrations of lipids measured in bee pollen could be arranged in the following descending order: autumn > spring > winter > summer. The highest values (%) of lipids (5.45), linolenic acid (C18:3) (20.81), stearic acid (C18:0) (20.06), linoleic acid (C18:2) (13.85), arachidic acid (C20:0) (6.59), and the sum of C18:0, C18:1, C18:2, and C18:3 (72.68) were obtained from bee pollen harvested during autumn. The maximum values (%) of oleic acid (C18:1) (36.63) and palmitic acid (C16:0) (25.91) were found in bee pollen harvested during summer. The highest concentrations (%) of behenic acid (C22:0) (11.18) and lignoceric acid (C24:0) (3.74) were found in bee pollen harvested during winter. Bee pollen harvested during spring ranked second in its lipid (5.05%), oleic acid (33.24%), palmitic acid (19.72%), linolenic acid (16.48%), stearic acid (12.20%), arachidic acid (5.54%), behenic acid (2.80%), and lignoceric acid (1.17%) concentrations.The data presented in The highest UFA values (61.51%) and the highest UFA/SFA ratio (1.60) were found in bee pollen harvested during summer, followed by bee pollen harvested during spring, which contained UFA and UFA/SFA ratio values of 58.47% and 1.41%, respectively. The highest concentration of EFA (34.66%) was found in bee pollen harvested during autumn, followed by an EFAs concentration of 25.26% measured in bee pollen harvested during spring. The highest concentration of SFAs (47.82%) was found in bee pollen harvested during winter, followed by an SFA concentration of 47.36% measured in bee pollen harvested during autumn .p < 0.05 and 0.01, respectively). Similarly, stearic acid was positively correlated  with linoleic acid, linolenic acid, and arachidic acid, and linolenic acid and arachidic acid were positively correlated with one another . Palmitic acid and linoleic acid were negatively correlated with behenic acid and lignoceric acid .The data shown in Rapeseed, sunflower, summer squash, date palm, and alfalfa were the main floral pollen resources in the Al-Ahsa Oasis in eastern Saudi Arabia. These floral resources contribute approximately 93.97% to 94.76% of the yearly collected bee pollen ,36. All The high lipid levels measured in autumn- and spring-harvested bee pollen were related to the relative abundances of pollen collected from summer squash and sunflower in autumn and from rapeseed, summer squash, and sunflower in spring. Bee pollen collected from rapeseed, sunflower ,21,50, ap < 0.01) differed among seasons and were dependent on the harvesting season. In the current study, oleic acid, palmitic acid, and linolenic acid were the most abundant FAs found in the bee pollen harvested during spring, summer, and winter. Similar results were found in Saudi bee pollen [The mean concentrations of fatty acids in the tested bee pollen significantly  than in summer (71.54%) and spring (70.67%), while the lowest value was reported in winter (63.40%). The sums of the C18:0, C18:1, C18:2, and C18:3 concentrations obtained in the current study were relatively similar to the corresponding concentrations measured in bee pollen collected from Saudi Arabia [The sum of the Ci Arabia  but werei Arabia .p < 0.01) differed among the seasons and were dependent on the harvesting season. The SFA concentrations in the studied bee pollen were higher in winter and autumn than in spring and were lowest in summer. The high UFA concentrations measured in the summer-harvested bee pollen were related to the relative abundance of alfalfa pollen, which has been reported to be a rich source of oleic acid [The SFA, UFA, and EFA concentrations and the SFA/UFA ratio values measured in bee pollen significantly  in the analyzed bee pollen were higher in autumn and spring than in summer and were lowest in winter. The high EFA concentrations recorded in bee pollen harvested in autumn were due to the relative abundances of summer squash- and sunflower-harvested pollen in this season; sunflower pollen ,20,56 anWe found seasonal variations in the lipid and FA contents of bee pollen. Oleic acid, palmitic acid, and linolenic acid were the dominant FAs observed in the analyzed bee pollen. The pollen loads collected during spring ranked second, with high concentrations of oleic, palmitic, linolenic, stearic, arachidic, behenic, and lignoceric acids, high concentrations of EFAs and UFAs, and a high UFA/SFA ratio value; spring-harvested pollen ranked first in the amount of harvested pollen loads. We recommend harvesting pollen loads during spring and summer to feed honeybee colonies during the periods of scarcity and supplementing bee pollen as a healthy, nutritious food for humans.


Primary liposarcoma arising from the liver is exceedingly rare. There have been very few reports documenting primary hepatic liposarcoma, especially of the pleomorphic subtype. Surgery is currently the only established treatment method, and the prognosis remains poor. In this report, we present an unusual case of hepatic liposarcoma of the pleomorphic subtype with literature review. In addition, we discuss theories regarding pathogenesis and the pathological and clinical features of primary hepatic liposarcoma to better outline this rare entity.An asymptomatic 65-year-old female was found to have a right hepatic mass on a computed tomography scan 2 years after surgical resection of the left adrenal gland and kidney for adrenocortical carcinoma. Laboratory examinations were unremarkable. Magnetic resonance imaging demonstrated a 16-mm mass in the right hepatic lobe. Adrenocortical carcinoma metastasis was suspected. Laparoscopic partial hepatectomy completely removed the tumor with clear margins. Macroscopically, the surgical specimen contained a nodular, yellow–white mass lesion 20 mm in diameter. On pathologic examination, pleomorphic, spindle-shaped tumor cells containing hypochromatic, irregularly shaped nuclei of various sizes formed fascicular structures. Scattered lipoblasts intervened in varying stages. Mitotic cells were frequent. Ki-67 labeling index was 15%. Immunohistochemically, the tumor cells were diffusely positive for vimentin and focally positive for CD34 and alpha-SMA; lipoblasts were focally positive for S-100. Tumor cells were nonreactive for SF-1, inhibin alpha, desmin, HHF35, HMB45, Melan A, MITF, c-kit, DOG1, cytokeratin AE1/AE3, h-caldesmon, STAT6, CD68, MDM2, CDK4, c17, DHEAST, 3BHSD, CD31, Factor 8, and ERG. From these findings, primary hepatic liposarcoma of pleomorphic subtype was diagnosed. The tumor recurred intrahepatically 3 years later, and the patient died 5 months after recurrence.In our report, we discussed the rarity, theories regarding pathogenesis, and a review of the literature of this atypical condition. To the best of our search, this is the 14th case of primary hepatic liposarcoma and the 2nd case of the pleomorphic subtype reported throughout the world. Further research regarding the etiology of this unusual clinical entity is warranted to establish effective diagnostic and management protocols. Liposarcoma is a rare malignant adipocyte tumor derived from mesenchymal cells. Representing approximately 15–20% of all soft tissue tumors, liposarcoma usually develops in the extremities and retroperitoneum [In this report, we present a rare case of primary pleomorphic liposarcoma originating from the liver with literature review. Due to the particular rarity of the pleomorphic variety, very few cases have been reported thus far. In addition, we discuss theories regarding pathogenesis and the pathological and clinical features of primary hepatic liposarcoma to better outline this rare entity.A 65-year-old Japanese woman was found to have a heterogeneous, ring-enhancing, 11-mm mass in the right hepatic lobe on follow-up contrast-enhanced abdominal computed tomography (CT) 2 years after surgical resection of the left adrenal gland and kidney for left-sided adrenocortical carcinoma. Medical and family histories were negative for hepatic disorders. The patient reported no symptoms and was subsequently referred to our department for further evaluation and treatment.On admission, physical examination revealed a well-appearing woman in no acute distress. Laboratory examinations were unremarkable. Serologic testing for hepatitis B and C was negative. Alpha-fetoprotein and des-γ-carboxy prothrombin levels were within normal limits. Dynamic contrast-enhanced T2-weighted images (T2WI) on magnetic resonance imaging (MRI) of the abdomen showed a 16-mm mass lesion with both cystic and solid, lipid-containing components , circulating MSCs, and hepatic progenitor cells (HPCs) are potential alternative origins Fig. . Hepaticsine qua non of liposarcoma diagnosis, dedifferentiated liposarcoma, myxoid liposarcoma, and myxoid pleomorphic liposarcoma were also differential diagnoses. Other differentials included epithelioid gastrointestinal stromal tumor (GIST), solitary fibrous tumor (SFT), and angiosarcoma due to positive staining for CD34. Adrenocortical carcinoma metastasis was also considered from the patient’s past medical history. Immunohistochemically, the lipoblasts stained positive for S-100. The tumor cells were negative for desmin, HHF35, HMB45, Melan A, MITF, c-kit, DOG1, cytokeratin AE1/AE3, h-caldesmon, STAT6, CD68, CD31, Factor 8, and ERG, making diagnoses of PEComa, GIST, SFT, and angiosarcoma unlikely. The presence of lipoblasts further ruled out angiosarcoma. Negative staining of CDK4 and MDM2 ruled out well-differentiated liposarcoma and dedifferentiated liposarcoma. Furthermore, negative results for SF-1, inhibin alpha, c17, DHEAST, and 3BHSD ruled out adrenocortical carcinoma metastasis. Split-signal FISH results negative for significant DDIT3 signal splitting ruled against myxoid liposarcoma. Similarly, low split signals for CREB1 and ATF1 lowered the possibility of angiomatoid fibrous histiocytoma. Myxoid pleomorphic liposarcoma was ruled out from the lack of characteristic myxoid matrix and oval cells. From these pathological and immunohistochemical findings, the patient’s tumor was classified as a hepatic liposarcoma of pleomorphic subtype.In 2020, the WHO renewed its classification criteria for liposarcoma to recognize five distinct subtypes based on pathological features , 2. The Pleomorphic liposarcoma is an aggressive neoplasm with an overall poor prognosis. According to previous studies focusing on pleomorphic liposarcoma from the orthopedic field , 5, the A PubMed literature search yielded 13 previous cases of primary hepatic liposarcoma (Table We reported a case of pleomorphic liposarcoma originating from the liver. Liposarcoma arising from the liver is so rare that clinicians may struggle to reach a definitive diagnosis. Due to the high recurrence rate and highly malignant progression, further research regarding the etiology of this unusual and highly malignant clinical entity is warranted to establish effective diagnostic and management protocols.


N-acetylglucosamine pyrophosphorylase (UAP), UDP-glucose pyrophosphorylase , and UDP-sugar pyrophosphorylase (USP). The evolutionary history of the UDPGPs indicated that the members of UAP, USP, and UGP-B were relatively conserved while varied in UGP-A. Homologous sequences of UGP-B and USP were found only in plants. The expression profile of UDPGP genes in Oryza sativa was mainly motivated under jasmonic acid (JA), abscisic acid (ABA), cadmium, and cold treatments, indicating that UDPGPs may play an important role in plant development and environment endurance. The key amino acids regulating the activity of UDPGPs were analyzed, and almost all of them were located in the NB-loop, SB-loop, or conserved motifs. Analysis of the natural variants of UDPGPs in rice revealed that only a few missense mutants existed in coding sequences (CDSs), and most of the resulting variations were located in the non-motif sites, indicating the conserved structure and function of UDPGPs in the evolution. Furthermore, alternative splicing may play a key role in regulating the activity of UDPGPs. The spatial structure prediction, enzymatic analysis, and transgenic verification of UAP isoforms illustrated that the loss of N- and C-terminal sequences did not affect the overall 3D structures, but the N- and C-terminal sequences are important for UAP genes to maintain their enzymatic activity. These results revealed a conserved UDPGP gene family and provided valuable information for further deep functional investigation of the UDPGP gene family in plants.UDP glucose pyrophosphorylase (UDPGP) family genes have been reported to play essential roles in cell death or individual survival. However, a systematic analysis on UDPGP gene family has not been performed yet. In this study, a total of 454 UDPGP proteins from 76 different species were analyzed. The analyses of the phylogenetic tree and orthogroups divided UDPGPs into three clades, including UDP- N-acetylglucosamine pyrophosphorylase (UAP), UDP-glucose pyrophosphorylase (UGP), and UDP-sugar pyrophosphorylase (USP). UAP prefers N-acetylglucosamine-1-P (GlcNAc-1-P) and N-acetylgalactosamine-1-P  as substrates  is a big gene family with three groups, UDP-bstrates . UGP is bstrates . For USPbstrates . The UDPthaliana , survivathaliana , and micthaliana , as wellthaliana .Saccharomyces cerevisiae), and loss-of-function mutant (uap1Δ) exhibited aberrant morphology including swelled or lysed  were identified belonging to UGP clades in A. thaliana with ATUGP1 predominantly expressed in most tissues (atugp1 or atugp2) did not show any deficiency, while an atugp1/atugp2 double mutant exhibited extreme deficiency in plant growth and male sterility. Further experiments showed that the destruction of the callose wall around microspores at the tetrad stage gives rise to abnormal development of pollen, resulting in male sterility in the double mutant (ATUGP1) regulates fumonisin B1-induced programmed cell death (UDP-glucose pyrophosphorylase 3 (AtUGP3) was reported in A. thaliana, which played a key role in sulfolipid biosynthesis  were reported in potatoes , and a knockout mutant of the USP gene exhibited an abnormal development in pollen, resulting in sterility  and 0 to 4 (USP), while the members in UGP (UGP-A and UGP-B) varied greatly, spanning from 0 to 14 (G. raimondii). Specifically, we did not find any full-length UAP genes in Kalanchoe fedtschenkoi or USP genes in Volvox carteri. In addition, Micromonas sp. RCC299, Micromonas pusilla, and Ostreococcus sp. Lucimarinus had no UGP (UGP-A or UGP-B) genes  method. The phylogenetic tree with gene names and bootstrap values (UFBoot/SH-aLRT/aBayes) is shown in  studies . Then, w studies . The resB) genes .The calculated isoelectric point (pI) and molecular weight (MW) of each UDPGP sequence are shown in The loss or gain of introns leads to different gene structures, makes genes more complex, and acts as the foundation of gene evolution . PreviouOryza sativa. Three OsUGP genes  posited on chromosome 9, chromosome 2, chromosome 3, respectively. Synteny analysis of the UDPGP family in rice showed that collinearity blocks between UDPGP members only existed in clade UAP (OsUAP1 and OsUAP2) and located on chromosome 8 and chromosome 4. Furthermore, the genome of O. sativa only contains a single OsUSP gene located on chromosome 6. The similarity of OsUAP1 and OsUAP2 indicated that these two genes originated from duplication. Among the five chromosomes in Arabidopsis, only chromosome 4 carried no UDPGP genes, and others had one or two UDPGP genes (ATUGP genes (ATUGP1 and ATUGP3), and chromosome 5 contained ATUSP and ATUGP2 genes at both arms. Besides, chromosome 1 and chromosome 2 separately hold GlcNA.UT1 (ATUAP1) and GlcNA.UT2 (ATUAP2) genes. In addition, we also detected duplication events, which were similar to O. sativa, in A. thaliana. The ATUAP1 and ATUAP2, ATUGP1 and ATUGP2 were two paired collinearity genes detected by McScanX, while no tandem gene pairs were identified in both O. sativa and A. thaliana. All these results showed that the members of UDPGP genes were stable and the expansions of the UDPGP gene family were only caused by segmental duplication.In the evolutionary history of the UDPGP gene family, the number of members was relatively stable, resulting in an elementary gene family. As shown in GP genes . ChromosO. sativa expression pattern in eight  different tissues (LOC_Os09g38030 (OsUGP1) was significantly higher than that of other gene members in the UDPGP family. Anther during flowering is the highest expressing tissue detected with OsUGP1. The LOC_Os02g02560 (OsUGP2) and LOC_Os01g15910 (OsUGP3) also belonged to the UGP clade in rice, whose expression levels were greatly lower than that in OsUGP1. For OsUGP2, anther (before and during flowering) and panicle (7 days before heading and 7 days after flowering tissues) in specific development stages exhibited higher expression levels than other tissues. Notably, OsUGP3 showed almost no expression in the seven tissues. Besides, for the UAP clade LOC_Os04g52370 (OsUAP2) and LOC_Os08g10600 (OsUAP1), the expression patterns were similar to each other and the expression level of OsUAP2 gene was slightly higher than OsUAP1 in some specific tissues, such as root and shoot from 7-day seedlings. In addition, for the OsUSP gene, a single member of the rice USP clade, the expression profile was ubiquitous in all tissues with a relatively low level. These results suggest that rice UDPGP genes can be expressed in various tissues to perform their roles with different expression levels as needed.The transcriptomic profile reflects the tissue-specific function. RNA sequencing data from Rice Expression Database (RED) were downloaded to analyze the UDPGP genes in  tissues . The resOsUAP1, OsUGP3, and OsUSP were generally upregulated in shoot tissue under the ABA and JA stress, and OsUSP was also upregulated in root tissue under the ABA and JA stress.Plant hormones, abscisic acid (ABA) and jasmonic acid (JA), are important regulatory factors involved in various biological processes, including cell death . The expOsUGP3  also showed increased expression trend in shoot tissue  were raised both in shoot (Cadmium (Cd) contamination has become a big issue in food safety, especially in rice . Under tOsUGP3 . The thrt tissue  under thin shoot  and rootin shoot .O. sativa under abiotic  and non-metal [phosphate (P)] ]  treatmenThe expression data above together implied that rice UDPGP genes may be involved in environmental endurance.Phylogenetic analysis of the UDPGP homologs yielded three big different clades and four distinct subgroups . In prevGLGTRLGCTGPK” displayed in all clades except UGP-B; this motif was corresponding to that of a previous study that reported a high essential signature motif “LX2GXGTX6PK”  was replaced by thymine (T) at the position of 712 bp in the CDS, resulting in the 238th amino acid changed from glycine (Gly) to cysteine (Cys) and thus lost function of UAP enzymatic activities for the OsUAP1 protein  and G210 (corresponding to G236 in OsUAP1) were substituted by alanine, the enzymatic activity of UAP was significantly reduced. In H. sapiens . These three sites corresponded to the K180, K329, and K402 in OsUGP1. In addition, the authors trimmed different lengths of N and C terminals to study the key role in regulating the activity. Results showed that the majority  largely reduced the activity except for the cut 32 amino acids in C terminal, indicating the importance of N and C terminals for catalytic activity. In S. tuberosum  in Cricetulus griseus  from Ser to Ala in OsUSP with 28.30%  of six UDPGP genes carried more than one transcript sequence from the rice annotation file. The OsUAP1 gene had three different types of mRNA sequences, OsUAP1.1, OsUAP1.2, and OsUAP1.3. The OsUAP1.1 transcript was the longest form with 489 amino acids, while the OsUAP1.2 transcript lacked exon sequence near 5’ untranslated region (5’ UTR), and the OsUAP1.3 transcript lost exons at 3’ UTR  spectroscopy was used to record the enzymatic reaction of OsUAP1.1, OsUAP1.2, and OsUAP1.3 in situ. In the time-gradient enzymatic progression at 60 min, forward conversion of GlcNAc-1-P (5.36 ppm) to UDP-GlcNAc (5.52 ppm) was observed with OsUAP1.1  to GalNAc-1-P (5.39 ppm) . The results showed that both OsUAP2.1 and OsUAP2.2 could catalyze the reaction from GlcNAc-1-P to UDP-GlcNAc, UDP-GlcNAc to GlcNAc-1-P, and UDP-GalNAc to GalNAc-1-P  , line 2,.39 ppm) , line 1,OsUAP1.2 , line 3,OsUAP1.3 , line 4 lNAc-1-P .OsUAP1 induces early leaf senescence  were mutated, corresponding to the 238, 203, and 238 in amino acid sequences (Gly to Cys), respectively  the inactive UDPGP gene isoforms are mistake alternative splicing; (2) the inactive UDPGP gene isoforms are under evolution for new functions; (3) the inactive UDPGP gene isoforms have effective but unknown functions, for example, competing with active isoforms to bind the enzymatic substrates to regulate the enzymatic activities of active isoforms; (4) lack of the C terminal could reduce the spatial block of the UDPGP, resulting in forming an inactive dimer in the plant . All aboin vitro enzymatic experiments showed that the OsUAP1 lost its catalytic activity without the N (OsUAP1.2) or C (OsUAP1.3) terminal, while OsUAP2.1 and OsUAP2.2 both maintained their catalytic activity with complete N and C terminals. The same results were also proven in in vivo transgenic experiments, where the early senescence phenotype of the mutant osuap1 could be rescued through overexpressing OsUAP1.1, OsUAP2.1, and OsUAP2.2, but not with OsUAP1.2 and OsUAP1.3. All above provide new insights into the evolution and function of the UDPGP gene family, which may lay a foundation to further investigate their molecular regulatory mechanisms in the plant.In the present study, the phylogenetic tree of the UDPGP gene family from 76 organism lineages divided this gene family into three clades, including UAP, UGP, and USP, and the UGP could be additionally separated into two subclades, UGP-A and UGP-B. This result was also supported by the diverse physicochemical features, gene structures, and motifs in different clades. Through scanning the UDPGP gene members in 76 species, we found that the number of the UGP-A is more variable among the clades, while UGP-B, UAP, and USP showed relatively conserved members, indicating the important role of these UDPGP genes. UDPGP genes significantly respond to cadmium, cold, ABA, and JA stresses in the shoot or root tissues in rice, while they do not exhibit obvious feedback to salinity, dry, flood, osmotic, and phosphate stimulation. Through MSA, we identified the key amino acids regulating the enzymatic activities of the UDPGP proteins, and many of them located in the NB-loop, SB-loop, and conserved motifs, demonstrating the key role of these structures. Alternative splicing may be a key mechanism to regulate the enzymatic activity of UDPGP genes. In the current study, 2 and Ensembl website3  Pfam-A.hmm was a manually corrected database and was downloaded from the Pfam website . Hmmscan software was used to identify the UDPGP domain with default parameters, and a total of 454 non-redundant primary protein sequences were retrieved for further analysis.Protein sequences, transcript sequences, genomic sequences, and GFF annotation files of 58 green plants, seven chlorophytes, six animals, three fungi, one kinetoplastid, and one bacterium were downloaded from Phytozomewebsite3 . UDPGP h4  information of the 105 full-length primary protein sequences from 16 representative plants was retrieved from the GFF annotation files and submitted to the TBtools  to visua TBtools . The pI/ TBtools  was usedO. sativa were downloaded from the Rice Expression Database7 and TENOR database8. The expression abundance with different treatments was transformed by log2RPK and visualized using TBtools.The UDPGP gene expression data of Amborella trichopoda, A. thaliana, Brachypodium stace, Citrus sinensis, Glycine max, Marchantia polymorpha, O. sativa, Panicum hallii, Physcomitrella patens, Prunus persica, Ricinus communis, Selaginella moellendorffii, Setaria italica, Solanum lycopersicum, Sorghum bicolor, and Triticum aestivum), including basal angiosperm, spikemoss, mosses, liverwort, monocots, and eudicots were selected to study the evolution of the UDPGP gene family , GST-OsUAP1.2 , and GST-OsUAP1.3 . The full-length CDS of the OsUAP2.1 and OsUAP2.2 isoforms were amplified using primers GST-OsUAP2 . PCR products were inserted into pGEX-6P-1 using the restriction enzyme sites BamHI and EcoRI. Then, the recombinant vectors were transferred into E. coli DH5α and sequenced to check if the constructions were correct. Expression and purification of the fused GST-UAP isoforms were performed using the same method as described , OsUAP1.2-OE , and OsUAP1.3-OE . The full-length CDSs of the OsUAP2.1 and OsUAP2.2 isoforms were amplified using primer OsUAP2-OE . PCR products were inserted into the binary vector pBWA(V)BU (reconstructed from pCAMBIA3300) using BsaI sites for the digesting-link one-step reaction. The recombinant vectors were transferred into E. coli DH5α and sequenced to check if the constructions were correct. Correct vectors were introduced into Agrobacterium tumefaciens EHA105 and then transformed into the osuap1 calli. Positive transgenic plants were confirmed using the phosphinothricin solution (20 mg/L) screening and then cultivated under natural conditions.The overexpression vectors of The original contributions presented in the study are included in the article/ZW designed the research. SL, HuZ, QW, CL, and ZW performed the research and analyzed the data. SL, HuZ, and ZW wrote the manuscript. TL, ZP, YL, HoZ, JL, YH, and ZW revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metaplastic breast cancer is a rare and often chemo-refractory subtype of breast cancer with poor prognosis and limited treatment options. Recent studies have reported overexpression of programmed death ligand 1 (PD-L1) in metaplastic breast cancers, and there are several reports of anti-PD-1/L1 being potentially active in this disease. In this case series, we present 5 patients with metastatic metaplastic breast cancer treated with anti-PD-1-based therapy at a single center, with 3 of 5 cases demonstrating a response to therapy, and one of the responding cases being a metaplastic lobular carcinoma with low-level hormone receptor expression. Cases were evaluated for PD-L1 expression, tumor infiltrating lymphocytes (TILs), DNA mutations, RNA sequencing, and T-cell receptor sequencing. Duration of the response in these cases was limited, in contrast to the more durable responses noted in other recently published reports. Metaplastic breast cancer (MBC) is a rare and aggressive subtype of breast cancer, comprising approximately 1% of all breast cancers, and is defined histologically as tumors that have epithelial differentiation into squamous and/or mesenchymal components, with multiple components often co-existing in the same tumor . The curst or 2nd line setting , measurable disease by RECISTv1.1, ECOG 0-1, and investigator-determined indication for paclitaxel or capecitabine in the 1 setting 4A, 5A setting , 19. TIL setting .When tissue was available, additional exploratory biomarker immune profiling was conducted. Cases 2, 4, and 5 were evaluated with a multiplexed immunofluorescence (mIF) panel as part of the clinical trial of pembrolizumab + chemotherapy in which they were enrolled . These c2) for quantification. The maximum possible number of non-overlapping regions of interest, as determined as areas with viable tumor and visible immune cells, were obtained for each slide. InForm software  was used according to manufacture instructions to segment and phenotype cells, with cells identified as cytokeratin-positive tumor cells, CD3-positive CD8-negative FoxP3-negative T-cells (Helper T-cells), CD3-positive CD8-positive T-cells (Cytotoxic T-cells), CD3-positive FoxP3-positive T-cells (Regulatory T-cells), and CD163-positive cells (Macrophages). PD-L1 quantitative immunofluorescence was also measured for each cell, which recent studies have found to be comparable to clinical PD-L1 scores  slides were stained and microwave treated in citrate buffer pH 6.0 to present cross-reactivity between antibodies. Tissue slides were incubated with DAPI as counterstain and coverslipped with VectaShield mounting media (Vector Labs). Whole slides were scanned and digitized at 10x magnification (PerkinElmer Vectra 3.0) for gross visualization of the tumor, with regions of interest scanned at 20x , MTOR, MYC, Nanog, NF2, CD274 (PD-L1), PI3K, PIK3RI, PTEN, and TP53  sequencing was performed in cases 1, 2, 4, and 5, and on n=21 non-metaplastic metastatic TNBC patients from the phase Ib trial. Peripheral blood mononuclear cells (PMBCs) were collected at baseline and at regular intervals during treatment, and T-cell DNA was extracted and submitted for deep sequencing using the immunoSEQ Assay (Adaptive Biotechnologies). T-cell richness was estimated by the nonparametric model iChao1 function, and clonality index was calculated as the square root of the Simpson’s diversity index.For the purpose of hypothesis generation, immune and genomic profiles were constructed for individual patients using the above biomarkers data. For each biomarker outcome, raw scores were converted into modified z-scores, based upon underlying median and median absolute deviations of the outcomes across a cohort of TNBC patients treated on the aforementioned phase Ib chemo-immunotherapy clinical trial. Because of the limited sample size, this analysis was conducted primarily for hypothesis generation and to identify possible outlier features of the case tumors, which could potentially assist with characterizing the unique clinical response profiles of each case in the series.The patient is a 63-year-old woman found to have a right breast mass on screening mammography, with biopsy showing a grade 3 invasive ductal carcinoma, ER-, PR-, HER2- . MRI additionally noted a small enhancing mass of the left breast, biopsy showing a concurrent grade 1 invasive ductal carcinoma with associated low-grade DCIS, ER >95%, PR 30%, HER2- (1+ IHC). She was treated with neoadjuvant therapy on the I-SPY trial with paclitaxel + ganetespib followed by doxorubicin + cyclophosphamide with a brief clinical response, followed by re-growth. She underwent bilateral mastectomy and sentinel lymph node biopsy, pathology consistent with metaplastic carcinoma, 3.1 cm x 2.8 cm with lymphovascular invasion, negative for perineural invasion, 2/2 intramammary lymph nodes involved with no extracapsular extension, 0/7 axillary nodes positive, 1/2 sentinel nodes with micro-metastatic carcinoma, no extracapsular extension, 0/11 additional axillary lymph nodes, and an RCB score of 3.835, class RCB-III   with capecitabine  . Per triPD-L1 expression and TILs were evaluated by a pathologist on pre- treatment and post- treatment biopsies. PD-L1 expression on tumor cells was 0% on both pre- treatment and post- treatment biopsies, but 10% and 40% respectively on immune cells. CPS measured 5 on the pre-treatment biopsy and 1.5 on post-treatment biopsies, both above the threshold for positivity of ≥ 1, but below the ≥10% threshold. TILs were 20% in the pre-treatment biopsy but decreased to 1% in the post- treatment biopsy.PIK3CA, TP53, PTEN, CDKN2A. In a comparison of RNA expression, there were no marked differences in expression within the TP53 or the RAS/MRK/ERK pathways, but PDK1 appeared less expressed within the PI3K pathway compared to other cases . Right axillary lymph node biopsy was positive for metastatic breast carcinoma. She received neoadjuvant dose-dense doxorubicin + cyclophosphamide, followed by paclitaxel, with decrease in the right breast mass but increase in an axillary dominant node on follow up ultrasound. She underwent lumpectomy and axillary lymph node dissection, with pathology showing a grade 3 invasive ductal carcinoma, 4.0 cm, with an additional 8 mm focus, 3/19 lymph nodes positive with the largest at 2.4 cm, negative for lymphovascular invasion. She received adjuvant radiation to the right breast. She later presented for follow up and reported increasing mid-back pain, with MRI of the T- and L-spine without evidence of metastasis to the spine, but found enhancing pulmonary lesions. CT chest noted bilateral lung lesions, with core biopsy showing an ER-, PR-, HER2- breast cancer with metaplastic features with focal chondroid differentiation.th line therapy with sacituzimab as of March 2021, with addition lines including eribulin, gemcitabine, cisplatin, and paclitaxel.She enrolled in the aforementioned phase Ib trial of capecitabine + pembrolizumab. Follow up CT scans at 12 weeks showed a partial response, with an overall shrinking of multiple lung nodules, while also noting growth of other smaller nodules . She had normal surveillance mammographies until November 2015 where she was found to have calcifications and possible distortion in the left upper outer breast. Biopsy found grade II pleomorphic invasive lobular carcinoma, ER 2%, PR-, HER2- . She had a left breast mastectomy with sentinel lymph node biopsy, with a 5 mm residual invasive lobular carcinoma, with additional foci ranging from 1-3 mm, grade II, with negative margins, and extensive lymphovascular invasion, 2/2 sentinel nodes positive. She received adjuvant cyclophosphamide, methotrexate and 5-FU.rd line compassionate use nivolumab with off-label bicalutamide as the patient had wanted to avoid further chemotherapy, and had not previously responded to ER-directed therapy. Bicalutamide was held after 2 weeks of treatment, with concerns for fluid retention and swelling. At 1 month follow up she had worsening skin lesions, but nivolumab was continued with the possibility of a flare reaction causing the exam findings rather than disease progression. 2 months into treatment skin lesions appeared to be crusting over, and at 4 months appeared to have a complete response , with androgen receptor staining positive in 30% of tumor cells. She received radiation, and then was started on fulvestrant + palbociclib, but had disease progression. She then started on exemestane + everolimus, but again had progressing skin lesions. She was then started on 3PIK3CA, TP53, AKT1, CDH1, KMT2D. Further genomic and immunoprofiling was unavailable for this case, as this patient was not a part of the clinical trial.PD-L1 expression on tumor cells was 2% of pre-treatment and 0% of post-treatment tumor cells were positive for PD-L1, compared to 50% of both pre-treatment and post-treatment immune cells. CPS was above the threshold for positivity of ≥ 1, and a higher threshold of ≥ 10 in the pre-treatment sample with a CPS of 10, though only above the ≥ 1 threshold in the post-treatment sample with a CPS of 3. TILs were scored as 30% in the pre- treatment and 15% in the post-treatment samples. DNA mutations included The patient is a 60-year-old woman who presented with a painful large left breast mass. Biopsy of the left breast showed grade 3 invasive ductal carcinoma with focal spindle cell features, also noted on left axillary biopsy, ER-, PR-, HER2- . She received 4 cycles of neoadjuvant dose dense doxorubicin + cyclophosphamide with minimal response, followed by 4 cycles of carboplatin + weekly paclitaxel with some response. She underwent a left modified radical mastectomy, with pathology showing a 4.4 cm grade 3 IDC with metaplastic features, and extensive lymphovascular invasion, clear surgical margins, and 4/7 axillary lymph nodes involved with extranodal extension. Prior to receiving adjuvant radiation, a subcutaneous nodule was found inferior to her mastectomy incision, with excisional biopsy showing 3 foci of recurrent/residual IDC with sarcomatoid features, with one focus extending beyond the excisional margin. She received adjuvant radiation, and a subsequent PET scan and brain MRI were without evidence of residual disease. She then presented with left arm swelling, CT chest, abdomen, pelvis found enlarged lymph nodes in the neck and chest, multiple pulmonary nodules, small hypodensities in the liver measuring less than 5 mm, and sclerotic-appearing lesions in the manubrium. A brain MRI and bone scan showed no evidence of metastases. An ultrasound-guided FNA of a neck nodule on the right showed extensive necrosis and degenerated atypical cells, consistent with a necrotic carcinoma.She was enrolled in the same phase Ib trial of capecitabine + pembrolizumab. Follow up imaging at 12 weeks noted a mixed response with growth of some nodes and regression of others, but she did have a new bony metastasis at T11 and was taken off of the trial.th lowest among the 19 evaluable cases. CD3+ Helper T-cells and CD8+ Cytotoxic T-cell counts were similar to non-metaplastic cases . Staging CT scan revealed a large left breast mass measuring 13.6 cm with a large left axillary node measuring 7.4 cm, numerous bilateral pulmonary metastasis, a suspected metastatic pancreatic neck mass measuring 1.8 cm, and a soft tissue lesion surrounding the right 10She received paclitaxel  with pembrolizumab (200 mg IV every 3 weeks). Following initiation, she had a mild infusion reaction to paclitaxel, but was maintained on therapy with dexamethasone pretreatment. The patient felt her breast mass shrank initially, but on follow up appointment prior to cycle 3, her mass appeared larger and repeat CT of the chest, abdomen, and pelvis showed progressive disease at multiple foci with a new pathologic fracture of the L-spine. She received palliative radiation to her spine and was taken off the trial and started on a DAE regimen . She developed disease progression and subsequently enrolled in hospice.TP53 and MYC. No significant patterns were noted in RNA expression in the TP53 and RAS/MEK/ERK pathway, though ERK2 was significantly lower in the RAS/MEK/ERK pathway. In the PI3K pathway, PIK3CA and PIK3R1 were relatively lower, but no pattern of reduced expression was noted in the rest of the pathway  in an attempt to identify the unique differences in each metaplastic case against TNBC, rather than a cohort that would include other metaplastic cases. Evaluation of immune cells by mIF demonstrated overall lower median raw cell counts across regions of interest in the metaplastic cases compared to the median of non-metaplastic TNBC cases. No obvious outliers were noted in comparison to non-metaplastic TNBC. Cases 2, 4, and 5 had positive z-scores in comparing CD3+ Helper T-cells to non-metaplastic TNBC, but all scores were <1 Table 4.SOX8, CIC, COL9A3, ZFAND1, UBE2W, C2orf40, ENY2, RBM39, TGS1, DPY19L4, CLEC18A, ACAN, SLC25A32, VIRMA, IGF2, NOTUM, WWP2, NPIPB11, UPK1B, GABPB1, NR4A1, SLC25A42, FBXO25. RNA expression in pathways of interest in MBC are further presented in Comparison of RNA sequencing did not demonstrate significant differences between metaplastic and non-metaplastic cases in multiple genes of interest, but did note multiple outlier genes, with an arbitrary cutoff of a modified z-score >3 in 2 or more metaplastic cases selected to identify possible outliers: TCR sequencing did not find significant changes in T-cell diversity by richness or clonality at baseline or during treatment between metaplastics and non-metaplastics. Evaluating the clonotype structure, metaplastics as a group vs non-metaplastics did not have significant differences in the amounts of higher frequency or lower frequency clones Figure 9Our case series provides additional evidence of clinical activity of chemo-immunotherapy for MBC, a rare subtype of breast cancer for which limited outcomes data are available. In this series, we describe clinical responses in 2/4 cases treated with chemotherapy plus pembrolizumab. Of interest, we also report a fifth MBC case of a complete clinical response to nivolumab and bicalutamide. These data are supportive of previously published reports of clinical response in MBC. Adams reported a case of metastatic MBC with a large chest wall lesion that dramatically responded to nab-paclitaxel + pembrolizumab, with an ongoing response at 6 months , whereasOne notable observation from our series is that clinical responses were less durable than previously reported in published case reports, with progression free survival (PFS) of 5.3, 5.7 and 8.0 months for Cases 1, 2 and 3 respectively. Of note, the non-metaplastic TNBC responders (n=8) in the same trial as Cases 1 and 2 had an average PFS of 6.9 months, arguing that duration of response to chemo-immunotherapy in metaplastic breast cancer may not appreciably differ from non-metaplastic TNBC. However, notably, Case 3 which had the longest PFS was an ER+ tumor, treated with bicalutamide in addition to anti-PD-1 therapy, and not a TNBC, limiting direct comparisons.A classic histologic trademark of MBC is intralesional heterogeneity, with the potential for having multiple regions of the tumor exhibiting distinct histologic features. In a recent analysis, it has also been suggested that intralesional histologic heterogeneity may reflect underlying genomic heterogeneity , 29. We ZEB1, a repressor of E-cadherin and Yes-associated protein . This study also found higher PD-L1 expression in tumor cells of MBC (44). In contrast, MBC cases had low PD-L1 expression Figure 7Three patients demonstrated a response to therapy, albeit limited in duration. One responding patient exhibited low-level ER expression and pleomorphic lobular features, whereas the other cases were triple negative breast cancer. Responses were observed in tumors with intermediate PD-L1 expression (CPS 1-10). The aggressive nature of MBC and unmet need for effective palliative options, support further investigation of the role of anti-PD-1/L1 in PD-L1-intermediate MBC is warranted.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Providence Portland IRB and Cedars IRB. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.IK – data analysis, manuscript writing. VR – data analysis. BB – data analysis. BC – data analysis. YW – data analysis. MM – data analysis. ZS – data analysis. WR – data analysis. KS – data analysis. RB – treatment of study subjects. HM – treatment of study subjects. DP – treatment of study subjects, manuscript writing and review. All authors contributed to the article and approved the submitted version.WR: Research support from Galectin Therapeutics, Bristol Myers Squibb (BMS), Merck, Tesaro/GlaxoSmithKline, MiNA Therapeutics, Inhibrx, Veana Therapeutics, Aeglea Biotherapeutics, Shimadzu, OncoSec, and Calibr. Patents/Royalties: Galectin Therapeutics. Advisory Boards: Nektar Therapeutics, Vesselon. DP: Received advisory board honoraria and institutional research funding support from Brooklyn ImmunoTherapeutics, Bristol-Myers Squibb, and Merck Laboratories. DP receives speaker bureau honoraria from Genentech and Novartis. Unrelated to this work, DP has received additional advisory board honoraria from other entities. RB: Consulting fees: Genomic Health, Astra Zeneca, Genentech; Institutional research funding: Seattle Genetics, Ichnos Biosciences, Merck; Honoraria for speakers bureau: Genentech, Seattle Genetics. HM: Consulting or Advisory Role - AstraZeneca; Bristol-Myers Squibb; Daiichi Sankyo; Genentech; Genomic Health; Immunomedics; Lilly; Merck; Pfizer; Puma Biotechnology; Puma Biotechnology; Seattle Genetics, Research Funding - Bristol-Myers Squibb (Inst); Lilly (Inst); Merck, (Inst); ZIOPHARM Oncology (Inst), Travel, Accommodations, Expenses - Amgen; AstraZeneca; Bristol-Myers Squibb; DAVA Pharmaceuticals; Genentech; Immunomedics; Lilly; Merck; Pfizer; Puma Biotechnology; Spectrum Pharmaceuticals, Other Relationship - Genomic Health; Lilly.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CCN5/WISP2 is a matricellular protein, the expression of which is under the regulation of Wnt signaling and IGF-1. Our initial characterization supports the notion that CCN5 might promote the proliferation and survival of pancreatic β-cells and thus improve the metabolic profile of the animals. More recently, the roles of endogenous expression of CCN5 and its ectopic, transgenic overexpression on metabolic regulation have been revealed through two reports. Here, we attempt to compare the experimental findings from those studies, side-by-side, in order to further establish its roles in metabolic regulation. Prominent among the discoveries was that a systemic deficiency of CCN5 gene expression caused adipocyte hypertrophy, increased adipogenesis, and lipid accumulation, resulting in insulin resistance and glucose intolerance, which were further exacerbated upon high-fat diet feeding. On the other hand, the adipocyte-specific and systemic overexpression of CCN5 caused an increase in lean body mass, improved insulin sensitivity, hyperplasia of cardiomyocytes, and increased heart mass, but decreased fasting glucose levels. CCN5 is clearly a regulator of adipocyte proliferation and maturation, affecting lean/fat mass ratio and insulin sensitivity. Not all results from these models are consistent; moreover, several important aspects of CCN5 physiology are yet to be explored. In recent years, we have reported a significant induction by IGF-1 of the expression of matricellular protein CCN5/WISP2 and its in vitro effects on promoting pancreatic β-cell proliferation and survival . Since tgrowth factors through their interactions with cognate receptors [The CCN/WISP family, which consists of six matricellular proteins, regulates development, cell adhesion and proliferation, extracellular matrix (ECM) remodeling, inflammation, and tumorigenesis. Most of these proteins contain four functional domains : the IGFeceptors ,13,14, Ceceptors .While searching for novel growth factors to promote the proliferation and survival of pancreatic islets, we revealed the expression of CCN5/WISP2  in resting pancreatic β-cells and its robust inductions by IGF-1, and further reported that recombinant human protein (rhCCN5) promotes mouse β-cell proliferation and survival in vitro .www.proteinatlas.org/ENSG00000064205-WISP2/tissue, accessed on 9 December 2021), CCN5 is a secreted plasma protein that is highly expressed in the proximal gastrointestinal tract, male and female reproductive tissues , and adipose tissue. Perhaps more accurately, Genotype-Tissue Expression (GTEx) project is an ongoing effort to build a comprehensive resource to study tissue-specific gene expression and regulation. From there, an expression profile of human CCN5/WISP2 can be retrieved, as shown in Before assessing its normal physiology, it is necessary to know where  CCN5 is normally expressed and how the expression might be regulated . It has been reported that CCN5 is expressed throughout murine embryonic development in most organs and tissues ,18. Accoheart tissue revealed CCN5 expression throughout the cytoplasm of the ventricular myocardium, in the atria and in the valves, and in most nuclei in the myocardium. In the lung, CCN5 was observed in the cytoplasm of alveolar and bronchiolar epithelial cells, endothelial cells, and smooth muscle cells, and also in the nuclei of many cell types [nuclear localization. All layers of mouse stomach wall and duodenum also displayed CCN5 staining in the cytoplasm. In mice, using IHC, CCN5 is expressed in the cytoplasm of acinar cells through the pancreas; but there was no nuclear staining [adipose protein secretome highlights CCN5 as a key regulator of obesity and ECM interactions [Immunohistochemical (IHC) analysis of mouse ll types . In bothstaining . In humaractions . Indeed,ractions .testis, with very high expression in peritubular cells and Leydig (testosterone-producing) cells. Numerous genes are expressed in mouse testis, yet most of them have not been studied for their involvement in spermatogenesis and sperm function. Analyzing the phenotype of CCN5-knockout mice might provide insight into the function of CCN5 in male reproduction. In rat and mouse ovaries, CCN5 is expressed in all cell types including stromal cells, thecal cells, granulosa cells, and oocytes [uterus in the smooth muscle, glandular epithelium, and the endometrium [Furthermore, CCN5 is highly expressed in the  oocytes . Previouometrium , and ratometrium . In the ometrium . In addiometrium ,23.As expected from previous reports, CCN5/WISP2 gene deficiency has been associated with mild obesity, insulin resistance, hyperglycemia, and lipotoxic cardiomyopathy . In a seelevated TGF-β/Smad3 signaling activities. Future experiments will be required to directly establish this mechanism.First, CCN5-knockout mice fed the normal chow diet (NCD) have been shown to exhibit mild obesity in comparison to wild-type littermates, despite no significant change in food intake . When feadipogenic genes and transcription factors, such as sterol regulatory element-binding protein (SREBP1), CCAAT/enhancer-binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor-gamma (PPARγ), and activating protein 2 (aP2) [Alongside obesity, the mass of subcutaneous and perirenal white adipose tissues (sWAT and pWAT) and of the heart was significantly increased in NCD-fed CCN5-knockout vs. wild-type mice . The inc 2 (aP2) . When ac 2 (aP2) . In the  2 (aP2) . With th 2 (aP2) . These rCellular hypertrophy was also exhibited in the heart of NCD-fed CCN5-knockout mice, with an increase in the mRNA expression of the hypertrophy-associated genes myosin heavy chain beta (β-MHC) and skeletal actin . HFD-fedoxidation-associated genes (CPT1β and MCAD) and glycerol-3-phosphate acyltransferase (GPAT), which is associated with triglyceride synthesis [accumulation of fat. Perhaps associated with that, NCD-fed CCN5-knockout mice also exhibited fibrosis in the heart, particularly in interstitial and perivascular areas. This was confirmed through collagen deposition in those areas and an increase in the expression of fibrosis-related genes  and inflammation-related genes (F4/80/CD3 and CD11c) [While NCD-fed CCN5-knockout mice exhibited lipid accumulation in the heart, they also showed an increase in the mRNA levels of lipid ynthesis . Althougd CD11c) . The lacd CD11c) . Furtherd CD11c) ,31.adipose tissues remains to be determined.Hence, CCN5 gene deficiency led to lipid accumulation in the heart, despite an apparent increase in lipid oxidation too. Cardiomyocyte hypertrophy may have occurred through a similar mechanism as in adipose tissues, involving increased differentiation of MPCs. Moreover, CCN5 knockout caused cardiac fibrosis and collagen deposition. The lack of CCN5 gene expression likely allowed the induction of a pro-fibrotic pathway due to elevated activities of the TGF-β/Smad3 signaling. On the other hand, whether this pathway is also involved in the induction of fibrosis in To further study the cellular mechanism, cardiomyocytes from NCD-fed CCN5 knockout-mice were isolated which exhibited mild insulin resistance. The latter was confirmed by reduced phosphorylation of AKT1 and GSK-3β following insulin administration relative to cardiomyocytes isolated from wild-type mice . This waHFD-fed CCN5-knockout mice did not exhibit such an increase vs. wild-type mice [Perhaps due to the mild obesity, lipid accumulation, and increased lipid oxidation, CCN5-knockout mice fed an NCD also showed mild hyperinsulinemia, hyperglycemia, and insulin resistance . On the ype mice .prolonged HFD (24 weeks) include mild obesity, further adipocyte hypertrophy, frank diabetes, insulin resistance, glucose intolerance, hyperinsulinemia, increased food and water intake, and further defect in cardiac function, perhaps due to lipid accumulation and cellular hypertrophy.As summarized in In summary, a systemic deficiency of CCN5 gene expression caused adipocyte hypertrophy, increased adipogenesis, and lipid accumulation, resulted in insulin resistance and glucose intolerance, which are further exacerbated upon HFD feeding. In addition, CCN5 deficiency caused cardiac fibrosis, cardiomyocyte hypertrophy, and lipid accumulation, leading to significant deficits in cardiac function. With little increase or no change in food intake, we would expect decreased energy expenditure in these mice, which has not been measured. Based on our reports, we also expect changes in pancreatic β-cell mass or function. It should be noted that these reports have never specified the sex of the animals [J.L. Liu et al. Unpublished observations). Nevertheless, current observations indicate that normal endogenous expression of CCN5 gene suppresses adipogenesis, but promotes/maintains cellular division, insulin sensitivity, and cardiac function. animals , while oCCN3/NOV, has also been reported to be an adipocytokine involved in obesity-associated insulin resistance (9). Excess CCN3 is associated with the induction of obesity, insulin resistance, and impaired adipocyte differentiation [decreased body weight and fat mass with an HFD, decreased adipocyte size, and significantly increased energy expenditure, glucose tolerance, and insulin sensitivity/signaling [Another member of the family, ntiation . In contignaling . IncreasIn pancreatic islets, CCN3 expression is upregulated in animal models of insulin resistance. CCN3 overexpression in vitro has been reported to disrupt β-cell function and proliferation, reducing cAMP levels, and decreasing glucose oxidation, thereby impairing insulin secretion . HoweverCCN4/WISP1 is a circulating factor that stimulates the proliferation of mouse and human β-cells [ β-cells . It is w β-cells .CCN2/CTGF, is essential for embryonic development of pancreatic islets [adult β-cells. CCN2-knockout mice die after birth and exhibit altered islet morphology with diminished β/α cell ratio [transient β-cell deficit at an early age [overexpression of CCN2 stimulated the replication of immature β-cells [Another family member, c islets ,36,37,38arly age . Combina β-cells ,37. BaseCCN2 is pro-fibrotic. Fibrosis is an excess deposition of extracellular matrix components including collagens causing the overgrowth, hardening, and/or scarring of various tissues. Normal tissues can be replaced with permanent scars. It is usually a result of chronic inflammatory reactions to persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, or other injuries [prevent fibrosis and can even reverse established cardiac fibrosis [In contrast to CCN5, however, injuries . Fibrosiinjuries . Indeed,fibrosis . In the CCN2 seems to be pro-fibrotic. Although not required for TGF-β-induced collagen or α-SMA expression in fibroblasts, CCN2 expression is necessary for the recruitment of pericyte-like cells in dermal fibrogenesis . Indeed,In clear contrast to the aforementioned findings from CCN5-knockout mice, adipocyte-specific CCN5 overexpression caused increases in lean body mass and insulin sensitivity, and cardiac hyperplasia .The human adipocyte fatty acid-binding protein (aP2) promoter was chosen to generate aP2-CCN5 transgenic mice because it can be activated by PPARγ and it has been used to specifically overexpress the canonical WNT ligand WNT10b in adipose tissues . FurtherTransgenic aP2-CCN5 mice overexpressing CCN5 exhibited no overall change in body weight when fed either a low-fat or high-fat diet (LFD or HFD) vs. wild-type littermates [When challenged with a period of HFD, aP2-CCN5 transgenic mice exhibited improved insulin sensitivity and decreased fasting glucose levels compared to wild-type mice [To confirm an increased level of lipogenesis, the serum levels of metabolically beneficial fatty acid esters of hydroxy fatty acids (FAHFAs) and their isomers, palmitic acid–hydroxy stearic acids (PAHSAs), were measured. Serum levels of 13/12- and 5-PAHSA were increased in HFD-fed transgenic vs. wild-type mice . The incThe findings of systemic CCN5 overexpression were associated with hyperplasia of cardiomyocytes, and white and brown fats . In thisDespite having a relatively stable fat mass, aP2-CCN5 mice exhibited smaller s.c. adipocytes and decreased serum leptin levels [In summary, the adipocyte-specific and systemic overexpression of CCN5 causes an increase in lean body mass, improved insulin sensitivity, hyperplasia of cardiomyocytes, and increased heart mass, but decreased fasting glucose levels. These results are mostly consistent with the opposite phenotypes exhibited in CCN5-knockout mice [out mice . Neverthout mice ,5. Moreo