Histology refers to the study of the morphology of cells within their natural tissue environment. As a bio-medical discipline, it dates back to the development of first microscopes which allowed to override the physical visual limitation of the human eye. Since the first observations, it was understood that cell shape predicts function and, therefore, shape alterations can identify and explain dysfunction and diseases. The advancements in morphological investigation techniques have allowed to extend our understanding of the shape-function relationships close to the molecular level of organization of tissues, as well as to derive reliable data not only from fixed, and hence static, biological samples but also living cells and tissues and even for extended time periods. These modern approaches, which encompass quantitative microscopy, precision microscopy, and dynamic microscopy, represent the new frontier of morphology. This article summarizes how the microscopy techniques have evolved to properly face the challenges of biomedical sciences, thus transforming histology from a merely qualitative discipline, which played an ancillary role to traditional "major" sciences such as anatomy, to a modern experimental science capable of driving knowledge progress in biology and medicine.

TGFβ plays a pivotal role in the pathobiology of myelofibrosis (MF) by not only promoting bone marrow fibrosis (BMF) but also by enhancing the dormancy of normal but not MF hematopoietic stem cells (HSCs). TGFβ has also previously been reported to inhibit normal megakaryocyte (MK) production (Bruno et al Blood 1998). TGFβ1 promotes the synthesis of collagen by normal human mesenchymal stromal cells (MSCs). Treatment of MSCs with AVID200, a potent TGFβ1/3 protein trap, significantly decreased MSC proliferation, phosphorylation of SMAD2, and collagen expression. Robust expression of pSMAD2 was observed in the absence of exogenous TGFβ in normal donor or MF-MKs, Addition of AVID200 to MKs decreased pSMAD2 without affecting total SMAD2/3 and led to increased numbers of MKs. Treatment of MF MNCs with AVID200 also led to increased numbers of progenitor cells with wild type JAK2 and a reduction of mutated colonies. A phase 1b trial of AVID200 (NCT03895112) was performed and completed in INT-2/high risk MF patients resistant/intolerant to ruxolitinib (rux); baseline platelet count of ≥ 25 x 10 9/L, and grade 2/3 BMF. Subjects received AVID200 intravenously on Day 1 of a 21 day cycle. Response was assessed by IWG/ELN criteria after 6 cycles of AVID200. Subjects attaining at least a CI or SD with a decrease in BMF by ≥1 grade, continued AVID200. We previously presented the results of the dose escalation study (Mascarenhas ASH 2020) demonstrating that AVID200 was well tolerated without dose limiting toxicities at 3 tested dose levels (Lots A and B) in dose cohorts of 180 mg (A), 550 mg (A)/70 mg (B), and 180 mg (B). Here we report updated safety and efficacy results of the phase 1b dose expansion stage at the two highest doses tested (70 mg (B) and 180 mg (B). Twenty-two subjects were enrolled (1 withdrew before receiving treatment) and 9 were treated with AVID200 in the dose escalation phase and 12 in the dose expansion phase [Table1]. Median time after rux discontinuation was 7.4 months (0.5-59.9). The most common mutations observed at baseline in this cohort included JAK2V617F (71%), TET2 (29%) ASXL1 (24%) and CALR (19%). (Fig 1) No DLTs were observed and Grade 3/4 AEs were observed in 16 (76.2%) subjects. Grade 3/4 non-hematologic AEs were observed in 8 (38.1%) subjects and included one subject in each case (epistaxis, mucositis, extraocular muscle paresis, fatigue, rash, duodenal hemorrhage, gastric hemorrhage, urinary tract infection, and syncope). Grade 3/4 hematologic AEs were anemia (6; 28.3%) and thrombocytopenia (2; 14.3%) [Table 2]. No fatal events were observed. The median number of cycles received was 5 (range 2 - 13) and 7 (33%) patients received more than 6 cycles. For dose levels 2-3 at cycle 7, a CI was attained in one subject at dose level 2 [anemia, spleen and TSS], 5 subjects had SD, 3 subjects had PD and two subjects with 10% and 15% blasts at screening developed MPN-BP while on study based on central review. Reasons for discontinuation by local PI included PD (n=8), lack of response (n=5), study completed (n=2), other (n=2), patient decision (n=1). Median % change in palpable spleen length was +10% (range -70% to +150%) and TSS change was -50% (-100% to +185.7%) The median platelet count at baseline was 114 x 10 9/L (range: 28-695) and 215 x 10 9/L (range: 66-263) after cycle 6 in 7 evaluable subjects (Fig 2A). Notably, 17 subjects had an increase in platelets from baseline during treatment and two subjects normalized their platelet counts. Maximum changes in platelets from baseline across all cycles was +63.8% [range -15.7%, +505.5%] (Fig 2B). Paired bone marrow biopsy pathology samples for 12 subjects were available for central review and showed no significant changes in BMF score or MK histo topography at end of treatment compared to baseline. All patients had elevated plasma levels of TGF β1, but not TGFβ2/β3 levels as detected by ELISA, which were dramatically reduced 21 days after the last dose of AVID200. AVID200 a TGFβ1/3 protein trap is well tolerated and clinical responses at cycle 7 of therapy in this advanced MF patient population were limited as judged by IWG/MRT response criteria. However, AVID200 therapy resulted in significant reduction in serum TGFβ levels and improvements in platelet counts indicating that TGF β1 plays a pivotal role in MF leading to thrombocytopenia which can be reversed with AVID200 therapy. We conclude that AVID200 may best be employed in combination therapy approaches in thrombocytopenic MF patients. Figure 1 Figure 1. Disclosures Mascarenhas: Constellation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Consultancy, Research Funding; Forbius: Research Funding; Genentech/Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Galecto: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prelude: Consultancy; Kartos: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Consultancy; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merus: Research Funding. Palmer: PharmaEssentia: Research Funding; Sierra Oncology: Consultancy, Research Funding; Incyte: Research Funding; CTI BioPharma: Consultancy, Research Funding; Protagonist: Consultancy, Research Funding. Kuykendall: Celgene/BMS: Honoraria; Pharmaessentia: Honoraria; Novartis: Honoraria, Speakers Bureau; Protagonist: Consultancy, Research Funding; Incyte: Consultancy; Abbvie: Honoraria; Blueprint: Honoraria. Mesa: Genentech: Research Funding; Promedior: Research Funding; Samus: Research Funding; Gilead: Research Funding; CTI: Research Funding; Abbvie: Research Funding; Sierra Oncology: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy; Pharma: Consultancy; CTI: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; AOP: Consultancy; La Jolla Pharma: Consultancy; Incyte Corporation: Consultancy, Research Funding. Rampal: Stemline: Consultancy, Research Funding; Memorial Sloan Kettering: Current Employment; BMS/Celgene: Consultancy; Abbvie: Consultancy; CTI: Consultancy; Novartis: Consultancy; Disc Medicine: Consultancy; Blueprint: Consultancy; Pharmaessentia: Consultancy; Incyte: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Constellation: Research Funding; Kartos: Consultancy; Sierra Oncology: Consultancy. Gerds: PharmaEssentia Corporation: Consultancy; Sierra Oncology: Consultancy; CTI BioPharma: Research Funding; Constellation: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; AbbVie: Consultancy; Novartis: Consultancy. Yacoub: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; ACCELERON PHARMA: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dynavex: Current equity holder in publicly-traded company; Cara: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Seattle Genetics: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau; Hylapharm: Current equity holder in publicly-traded company. Talpaz: Imago: Consultancy; Constellation: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Other: Grant/research support ; Celgene: Consultancy. Komrokji: Acceleron: Consultancy; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; Jazz: Consultancy, Speakers Bureau. Kremyanskaya: Astellas: Research Funding; Astex: Research Funding; Chimerix: Research Funding; Bristol Myers Squibb: Research Funding; Constellation: Research Funding; Protagonist Therapeutics: Consultancy, Research Funding; Incyte: Research Funding. Salama: Mayo Clinic: Current Employment, Other: Mayo Clinic had the contractual work for the central pathology review for this study and I was one of the reviewing pathologists; Constellation Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Hoffman: Kartos Therapeutics, Inc.: Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Novartis: Other: Data Safety Monitoring Board, Research Funding; AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding. OffLabel Disclosure: AVID200 is a TGFb trap and is in clinical testing for fibrotic diseases. It does not have an approved indication at this time.

We have investigated, by semiquantitative RT-PCR, the kinetics of activation of hematopoietic receptors and differentiation markers in partially purified murine hematopoietic stem cells (HSC) induced to differentiate in serum-free culture with combinations of growth factor (GF). The combinations of GF used sustained either multilineage [stem cell factor (SCF) + interleukin 3 (IL-3), or erythroid [SCF + IL-3 + erythropoietin (Epo)] or myeloid [SCF + IL-3 + granulocyte colony-stimulating factor (G-CSF)] differentiation. The GF receptor genes investigated were the alpha and beta subunits of the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin receptor, the G-CSF receptor, and c-Fms, the receptor for macrophage colony-stimulating factor (M-CSF). The expression of Gata1 and alpha- and beta-globin was investigated at the same time as a marker of erythroid differentiation. HSC were purified according to standard protocols, which include partitioning of lineage-negative bone marrow cells with the mitochondrial dye Rhodamine 123 (Rho) into Rho-dull (> or = 17% of which reconstitute long-term hematopoiesis in recipient mice) and into Rho-bright (which are as capable as Rho-dull of multilineage differentiation but do not permanently reconstitute the host). The following pattern of expression was observed: the alpha subunit of the IL-3 receptor clearly was expressed in both Rho-bright and Rho-dull cells at the outset, and its expression did not change over time in culture. The beta subunits of the IL-3 and GM-CSF receptor, the alpha subunit of the GM-CSF receptor, the Epo and G-CSF receptors and Fms barely were expressed in purified Rho-bright and Rho-dull cells, but their expression increased in cells cultured both in erythroid and in myeloid GF combinations. Gata1 was expressed maximally in Rho-bright cells but was below the level of detection in Rho-dull cells. Rho-dull cells expressed Gata1 when cultured both in erythroid and in myeloid GF combinations. In contrast, alpha- and beta-globin, which also were not expressed in the purified cells, were induced only in cells stimulated with Epo. These results indicate that the genes for all the GF receptors investigated (with the exception of the alpha subunit of the IL-3 receptor) are expressed at low levels, if any, in purified Rho-bright or Rho-dull cells, but are expressed in their progeny cultured either in erythroid or myeloid GF combinations. The expression of the Epo receptor, in particular, is activated both in erythroid (alpha- and beta-globin positive and in myeloid (alpha- and beta-globin negative) cells. Therefore, activation of the expression of the Epo receptor gene and activation of the erythroid differentiation program are two independent events in normal hematopoiesis.

Chronic idiopathic myelofibrosis (IM) is a chronic myeloproliferative disorder characterized by splenomegaly, a leukoerythroblastic blood picture, teardrop poikilocytosis, marrow fibrosis, osteosclerosis, marrow neo-vascularization, abnormal stem/progenitor cell trafficking and extramedullary hematopoiesis. The disease may eventually evolve into acute leukemia. This Philadelphia chromosome negative disorder is thought to originate from a somatic mutation at the level of the multipotent hematopoietic stem cell, the most visible consequence of which is a profound hyperplasia associated with increased proliferation but abnormal differentiation of the megakaryocytes (MKs). The pathobiology of the disease, however, involves not only abnormal hematopoietic stem/progenitor cells functions, but also a defective marrow microenvironment. The molecular nature of the genetic defect in IM and how this defect might induce so many pleiotropic consequences remains unknown. Many of the features of the human disease can be reproduced in mice by genetic alterations that induce MK abnormalities similar to those found in patients. Unfortunately, none of the mutations causing the disease in mice has been detected in the human disease. These animal models, however, allow one to dissect the patho-biological pathway that establishes the complex features of IM. Furthermore, these models also shed light on the cross-talk between stem/progenitor cells and microenvironment in normal hematopoiesis.

A mayor pathobiological role for interleukin 8 in the etiology of myelofibrosis has been suggested by observations indicating that megakaryocytes expanded in culture from these patients express great levels of interleukin 8 1 and that the plasma levels of this cytokine are predictive of poor prognosis 2. In preliminary experiments we demonstrated that the megakaryocytes from the bone marrow of the Gata1 low model of myelofibrosis express not only high levels of TGF-β, but also levels greater than normal of lipokalin-2, a known inducer of IL-8 production, and of CXCL1, the murine equivalent of IL-8. In addition, these megakaryocytes express also high levels of the CXCL1 receptors CXCLR1 and CXCR2 and the bone marrow from these mice express an CXCR1/CXCR2 activated signature. Using these data as a foundation, we tested here the effects of treatment of Gata1 low mice with the CXCR1/R2 inhibitor reparixin on the myelofibrosis phenotype expressed by this models. To these aim, Gata1 low mice (8-month old) were treated either with vehicle (3 males and 3 females) or with reparixin (formerly referred to as repertaxin) 3 (5 males and 5 females) for either 20 or 37 days. The drug was administered by minipumps implanted subcutaneously in the dorsal region set to deliver 7.5mg of drug/hr/Kg of body weight. The mice receiving the drug for 37 days had the minipumps replaced by day 17. The efficiency of drug delivery decreased over time since the plasma levels of reparixin were 13.90±4.18 and 6.71±4.18ug/mL at day 20 and 37, respectively (p<0.05).The drug was well tolerated with no death or change in body weight recorded over the period of observation. Since the results observed in males and females were similar, the data were pooled for statistical analyses. The treatment did not affect blood values (hematocrit (%): 34.32±3.87 vs 35.63±3.45 and 30.92±3.58, platelets: (x10 3/uL) 187.80±26.12 vs 181.30±53.30 and 99.83±71.92 and white cell counts (x10 3/uL): 2.78±0.55 vs 3.27±0.72 and 3.57±1.43, respectively, in vehicle and day 20- or day 37-reparixin treated mice). The treatment had also little effects on bone marrow (20.55±5.83 vs 22.24±0.85 and 21.68±6.49) and on spleen 141.40±29.04 vs 99.54±15.55 and 173.00±76.54) cellularity. However, the bones were reddish and their sections contained great numbers of erythroid cells, a sign of increased hematopoiesis. Great reductions in the fibrosis of the bone marrow and spleen was observed in mice that had been treated with reparixin compared to vehicle which were statistically significant by day 20 (day 20 bone marrow fibrosis 28.09±15.69 in vehicle and 4.54±0.45 in reparixin treated mice by Gomori, p<0.05; 19.30±7.86 vs 3.19±1.89 by reticulin, staining, p<0.05, respectively by Anova; day 20 spleen fibrosis 20.51±5.25 in vehicle and 10.85±3.82 in reparixin treated mice by Gomori, p<0.05; and 13.15±3.06 vs 6.13±2.34 by reticulin, staining, p<0.05, respectively). Of note when the levels of Gomori and reticulin fibrosis detected at day 20 and 37 in individual mice were inversely correlated with the plasma levels of reparixin observed in the same mice (Figure 1, p<0.01-0.05 by Pearson). Mechanistic insights on these results were provided by Immunostaining of marrow and spleen sections of vehicle and reparixin-treated mice indicating that the megakaryocytes from the reparixin-treated group express levels of TGF-β significantly lower than those expressed by the corresponding cells from vehicle while the levels of LCN-2, CXCL1, CXCR1 and CXCR2 expressed by the reparixin treated megakaryocytes are similar to that of the vehicle treated cells. These results indicate that inhibition of CXCL1 by reparixin, probably by reducing the abnormally high TGF-β content of the megakaryocytes, reduces fibrosis in Gata1 low mice and provide a preclinical rational to test this drug in patients with myelofibrosis. References: 1) Emadi S et al. Blood. 2005;105:464; 2) Tefferi et al, J Clin Oncol. 2011;29:1356; 3) Bertini R et al, PNAS 2004; 101:11791 Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Massucci: Dompe Farmaceutici Spa R&D: Current Employment. Brandolini: Dompe farmaceutici Spa R&D: Current Employment. Giorgio: Dompe farmaceutici Spa R&D: Current Employment. Allegretti: Dompe farmaceutici Spa R&D: Current Employment. Migliaccio: Dompe farmaceutici Spa R&D: Other: received funding for reserach .