#JAK2 Antibody

[bostorbio]

The JAK2 Antibody checks for blood levels of total endogenous JAK2 in the body. There was no evidence of cross-reactivity between members of the same family when tested in physiological conditions.

[garden-of-everything]

ABIM: Hematology

ABIM syllabus can be found here Let me know if you find any errors Sources: UWorld, MKSAP 16/17, Rizk Review Course, Louisville Lectures, Knowmedge (free version)

Hypoproliferative anemia

Aplastic anemia: - associated with autoimmune diseases, thymomas <-- need CT chest, Tx with resection - Tx all patients with PPx anticoagulation + iron + folic acid; if <40yo: stem cell transplant - associated with PNH: pancytopenia/hemolytic anemia with need for transfusions + Budd-Chiari/venous thrombosis/CVA/MI + morning hematuria/iron deficiency, associated with AML; Dx: flow cytometry shows lack of CD55, CD59; Tx: iron + Eculizumab to decrease need for transfusions (AE of Eculizumab is increased risk of meningococcal infections) +/- (if thrombus) warfarin; if <40yo: allogenic BMT Iron deficiency anemia: - decreased iron, ferritin, transferrin; increased TIBC, RDW - associated with gastric surgery, restless leg syndrome - colon cancer until proven otherwise - also seen in: (1) Celiac disease:  IBS Sx with iron deficiency that is unresponsive to oral supplementation; Dx: tissue transglutaminase Ab --> if neg: small bowel biopsy (2) Plummer-Vinson: esophageal webs, glossitis; associated with squamous cell esophageal carcinoma (3) Osler-Weber-Rendu/Hereditary Hemorrhagic Telangiectasia: lip/oral telangiectasias + epistaxis + +FOBT associated with hemoptysis/brain bleed - Tx iron deficiency: PO supplementation x6mo or IV iron gluconate (dextran is associated with anaphylaxis) Sideroblastic anemia: - ringed sideroblasts/basophilic stippling on PBS seen in EtOH, lead toxicity (Burton line on teeth, deposition in metaphysis, RTA type II (hypokalemia, glucosuria)), copper deficiency, and use of INH (from B6 deficiency), chloramphenicol, Linezolid - Tx hereditary sideroblastic anemia with pyridoxine/B6 Megaloblastic anemia:  MCV>100, hypersegmented PMNs, associated with EtOH use (1) B12 deficiency: peripheral paresthesias; associated with strict vegetarians and Crohn’s disease (terminal ileum disease), elevated methylmalonic acid > homocysteine (2) Pernicious anemia: anti-IF antibodies causing B12 deficiency; associated with autoimmune diseases and increased risk of gastric cancer (3) folate deficiency: no paresthesias; associated with pregnancy, EtOH, and Bactrim use; elevated homocysteine only Pure red cell aplasia:  associated with AIDS + Parvovirus B19; Dx: flow cytometry shows monoclonal CD57+ T-cells; Tx: IVIg and check for thymoma with CT chest

Hemolytic anemia

- PBS shows schistocytes (vs. dacryocytes/tear drop cells with 2 line involvement in Myelodysplastic syndrome) - appropriate reticulocyte response = >100,000/uL - Tx chronic hemolytic anemia with folate - Scleroderma renal crisis (HTN, AKI, MAHA):  Tx with ACEi (Captopril) even iff pregnant - MAHA in the setting of mechanical heart valve needs emergent TEE to check for leak/regurg Glucose-6-phosphate dehydrogenase deficiency: - remember food: fava beans, bite cells, Heinz bodies (red spots in RBCs; looks kind of like a pink boob with 1 or more red nipples) - associated with sulfa drugs, infection, and DKA --> hemolysis - has decreased glutathione levels Pyruvate kinase deficiency: Tx with PRN transfusions --> severe?: splenectomy Autoimmune hemolytic anemia:  Coomb’s/DAT positive (1) warm immunoglobulins: - IgG, DAT+ with spherocytosis - associated with autoimmune (SLE, UC), HIV, CLL - Tx steroids --> splenectomy --> refractory Tx: IVIg, AZT, cyclophosphamide, Rituximab (2) cold: - IgM (Mmm cold ice cream), occurs in cold temperatures - associated with malignancies, mono, mycoplasma - Tx: avoid the cold, Rituxan Microangiopathic hemolytic anemia:  Tx: plasma exchange +/- corticosteroids - Sx:  “FAT RN” (fever, anemia, thrombocytopenia, renal failure, neuro sx) - Dx: normal coags, D-dimer, fibrinogen (vs. DIC = elevated coags and D-dimer; decreased fibrinogen, platelets) *FYI:  ITP, TTP/HUS, and HELLP all have normal coags (1)  TTP:  primarily neurologic symptoms - increased vWF multimers/decreased ADAMSTS13 (NOT needed for diagnosis) - associated with tacrolimus, cyclosporine, plavix/ticlodipine, quinine - Tx: plasmapheresis *if >20 weeks pregnant, this is TTP and does NOT resolve with delivery (2)  HUS:  primarily renal symptoms - associated with E.coli/Shigella diarrhea and cyclosporine - Tx: discontinue cyclosporine!, supportive, plasmapheresis Hereditary spherocytosis: - jaundice from unconj/indirect hyperbilirubinemia, pigmented gallstones, splenomegaly, leg ulcers - PBS shows spherocytes and Howell-Jolly bodies (blue dot in RBC seen in splenectomy); DAT negative (vs. DAT+ in warm hemolytic anemia; see above) - Dx: osmotic fragility test --> flow cytometry - Tx: splenectomy Wilson’s: young patient with hemolytic anemia + psychosis + transaminitis

Hemoglobinopathies and thalassemias

- present as microcytic anemias: MCV <80; target cells Alpha thalassemia: has normal Hb electrophoresis --, -x = HbH: hemolysis, splenomegaly, Heinz body - --,-- = Barts / Hydrops fetalis (dies before birth) Beta thalassemia:  abnormal Hb electrophoresis - minor: elevated HbA2 (alpha 2 gamma 2) and HbF - intermedia:  elevated HbA2 only; Tx: intermittent transfusion +/- iron chelation if Fe>1000 - Major:  elevated HbF, decreased HbA;  Tx: splenectomy and allogenic stem cell transplant Hemoglobinopathy:  severe disease requires stem cell transplant

Leukocyte disorders

(1) AML:  t(15;17) - associated with PCV, Fanconi’s, Down’s, Klinefelter’s, CML, XRT/chemo, benzenes, MDS - presents acutely as sepsis (decreased PMNs, anemia, thrombocytopenia) - pallor, gingival hypertrophy (M5), fatigue, easy spontaneous bleeding/purpura (due to thrombocytopenia), and NO hepatosplenomegaly or lymphadenopathy -  M3 (Auer rods) associated with DIC Tx: ATRA *ATRA AE: after 1-3 weeks --> fever, leukocytosis, pulmonary infiltrates/hypoxemia; Tx: dexamethasone *prevent TLS (hyperkalemic paresthesia/weakness, hypocalcemic tetany, bronchospasm, AKI) with Allopurinol BEFORE chemo --> Rasburicase (2) CML: t(9;22)/BCR-ABL, decreased LAP - asymptomatic splenomegaly with elevated WBC (symptomatic when >200) with increased Eosinophils and Basophils - Tx: Imatinib/Gleevec (TKI) (3) ALL:  TdT, anterior mediastinal mass (thymoma <-- chest CT)/bulky mediastinal lymphadenopathy with bone pain and CNS involvement - increased blasts (>30%) - Tx: combo chemo (intrathecal if CNS involvement) +/- XRT if bulky disease --> stem cell (4) CLL:  B2microglobulin; CD5, CD23 B-cells; smudge cells - asymptomatic, lymphadenopathy, hepatosplenomegaly, lymphocytosis, thrombocytopenia - Tx: watchful waiting if asymptomatic; Bendamustine+Rituxan > Fludarabine + Bactrim PPx *AE:  Richter’s syndrome: transformation into aggressive large cell lymphoma *Evan’s syndrome: AIHA + ITP (5) Hairy cell:  older patient with pancytopenia, splenomegaly, dry fibrotic bone marrow - associated with PAN - Dx: flow-cytometry: CD11c, CD103+; +TRAP - Tx: Cladribine

Platelet disorders

*Rule of thumb:  transfuse if Plt <10 or if ICH/pulm hemorrhage <40-50; 1 bag of platelets corrects by 25k *give IVIg + steroids if pregnant with Plt <50 *Plt >50 = okay for surgery Idiopathic thrombocytopenic purpura:  diagnosis of exclusion - Dx: PBS, DAT+ (don’t order anti-Plt antibodies) - Tx (if symptomatic or Plt <15): steroids --> IVIg/Rhogam --> refractory: splenectomy, Rituxan Heparin-induced thrombocytopenia (HIT): - 4T’s: thrombocytopenia (2pts: >50%, 1pt: 30-50, 0pt: <30), timing (>5days, ?>5, <4), thrombosis (+, ?, -), no other cause (yes, maybe, no); score 6-8 = high, 0-3 low - Dx: PF4, SRA - Tx: stop Heparin (warfarin is NOT contraindicated!); switch to Argatroban Essential thrombocythemia: - “Jack et Vera are Buddies” (”et” in Latin is “and”): Jak2 mutations are associated with ET, polycythemia Vera, and Budd-Chiari” - JAK2, CALR, BCR-ABL, NPL1 - Plt >600 --> livedo reticularis, erythromelagia (painful red hands/feet, Tx: ASA), headache, vision changes - Tx: ASA + (if symptomatic) Hydroxyurea +/- (if TIA/CVA/MI/GIB) pheresis Other platelet dysfunction:  increased bleeding time; bleeding from small injuries, epistaxis, menorrhagia (1) vWF: increased PTT/low-normal VIII corrected with mixing study; Dx: vWF Ag; Tx: pre-dental DDAVP or for active bleed, recombinant factor VIII (2) Bernard-Soulier: X glycoprotein Ib --> thrombocytopenia (3) Glanzmann (~Abciximab/Eptifibatide): X gpIIb-IIIa --> normal platelet counts

Coagulation factor disorders and thrombotic disorders

Rule of Thumb: PeT PiTTbull - PT extrinsic pathway (VII, X) - PTT intrinsic pathway (VIII, IX, XI, XII) *mixing studies correct = deficiency --> present as bleeding into muscle/joint; Tx with DDAVP for mild disease or missing factor for active bleed *mixing studies don’t correct = (1) if there is bleeding: presence of factor antibody: Tx with recombinant FVII (2) if there is no bleeding:  antiphospholipid Ab

Coagulation factor deficiencies: - VII:  elevated PT (it’s pretty much the only one that isn’t elevated PTT), presents as ICH, Tx: rfVII - VIII/Hem A:  elevated PTT, bleeding into muscles/joints, no excessive bleed after minor cuts; Tx: DDAVP before dental work, fVIII - acquired VIII: elevated PTT that doesn’t correct, Tx: rFVII (<--yes, Tx is rf7, NOT 8) - IX/Hem B:  elevated PTT, bleeding into muscles/joints, no excessive bleeding after minor cuts; Tx: fIX - XI/Rosenthal: elevated PTT in Ashkenazi Jews; Tx: FFP prior to major surgeries - XII:  asymptomatic and totally benign elevated PTT - XIII:  coags look normal, but has severe post-op bleed; Dx: urease clot dissolves; Tx: Qmonthly FFP - acquired X/AL amyloid:  elevated INR with postural hypotension, macroglossia, heart failure, and proteinuria/kidney failure; Dx: serum/urine electrophoresis, free light chain assay, BMB, fat pad aspirate vs.  vWF: increased bleeding time, normal/elevated PTT that corrects with mixing stud - Dx: vWF Ag, vWF activy assay, VIII, level, subtype multimer study - Sx: gingival/mucocutaneus bleed, menorrhagia, easy bruising - Tx: DDAVP prior to dental work, active bleed: rfVIII

Thrombophilia:  DVT/PE’s (1) Antiphospholipid antibody syndrome: anti-beta2glycoprotein I Ab - blood clots, miscarriage; associated with SLE - apTT doesn’t correct with mixing study --> Dx: anticardiolipin IgG/IgM ELISA (2) Factor V Leiden: most common - Dx (check months after DVT): clotting assay (resistance to Protein C) --> genetic prothrombin G20210A mutation 

Porphyria

-morning hematuria, blistering photosensitive rash, abdominal pain, nausea/vomiting, HTN, tachycardia, psychosis, seizures all exacerbated by EtOH, smoking, stress, sulfa drugs - increased risk of HCC, lymphoma - Dx: elevated urine uroporphyrinogen (urine turns purple in sunlight) --> check for Hep C and hemochromatosis - Porphyria cutanea tarda: associated with HIV, Hep C; Tx: phlebotomy to decrease iron stores

Myeloproliferative disorders

Polychythemia vera and other erythrocytosis:  JAK2V617F, associated with Budd-Chiari, facial plethora, pruritus with hot baths - BMB is hypercellular, decreased EPO levels - Tx: ASA and phlebotomy to Hct <45 +/- Hydrea - also Tx hyperuricemia with allopurinol, pruritus with antihistamines Essential thrombocythemia:  JAK2, decreased EPO levels; associated with vWF disease (more info under Platelet Disorders above); Tx: ASA + Hydroxyurea +/- pheresis if TIA/CVA/MI/GIB Agnogenic myeloid metaplasia and myelofibrosis: - splenomegaly, dacryocytes, giant megakaryocytes/platelets, BM fibrosis - portal HTN - Tx: supportive (NOT splenectomy); if <60yo, stem cell transplant

Myelodysplastic syndrome

- cytopenia of 2 cell lines + tear drop cells + nucleated RBCs, elevated MVC - r/o B12 deficiency (PBS macrocytosis) - BMB shows ringed sideroblasts, Pseudo Pelger-Huetz cell (looks like 2-lobed PMN or cell wearing blue sunglasses) - Tx: Azacitidine to keep Plt >100k, Epo, GCSF; if young: stem cell transplant - 5q-subtype Tx (best prognosis): Thalidomide (AE: rash, peripheral neuropathy), Lenalidomide (AE: less neuropathy, but more decr PMN, thrombocytopenia)

Hematologic malignancies

Acute and chronic leukemias (see Leukocyte Disorders above) Hodgkin’s disease:  B-symptoms, non-tender contiguous nodes with Reed-Sternberg (owl-eye) B-cells, associated with EBV; Dx: full excision of lymph node + pan-CT + PET + BMB if Bsx or stage III/VI; Tx: ABVD + rads if same side of diaphragm --> screen for breast cancer 8 years after XRT or at 40yo Non-Hodgkin’s lymphoma:  multiple nodes with extranodal involvement, B>T cells, associated with autoimmune dz (Sjogrens: parotid MALT), HIV (1) Indolent: (a) Follicular: painless swelling in neck, armpit, groin; Tx: watchful waiting, but if symptomatic: XRT if limited, Bendamustine-Rituxan or R-CHOP if outside XRT field (b) MALT: Tx H.pylori (2)  Aggressive: (a) Diffuse large B-cell:  B-symptoms; Dx: biopsy, Tx: CHOP --> R-CHOP --> XRT (b) AIDs-associated lymphoma:  EBV in CSF --> primary cerebral lymphoma (c) Burkitt’s lymphoma:  African jaw mass/U.S.A. abdominal mass, associated with EBV, increased LDH, starry sky (3) Cutaneous T-cell (CD4): Sezary/Mycosis Fungoides:  plaque --> nodular lesions with cerbriform nuclei (epidermis: Pautrier microabscess) Plasma cell disorder / Multiple Myeloma: - “CRAB” hypercalcemia, renal injury, anemia, lytic bone lesions - >3gM protein, >10% plasma cells in BM, normal ALP, discrepancy between urine protein and urine dipstick (due to inability to detect light chains) - Dx: BMB, XR>bone survey; serum and urine electrophoresis Q6 months - Tx: (1) <75yo: Lenalidomide/Thalidomide + Dexamethasone --> stem cell transplant --> Bortezomib (2) can’t or >75yo: Melphalan + Prednisone vs. Smoldering MM: MM without Sx vs. Plasmacytoma: solitary lytic bone lesion; Dx: tissue Bx; Tx: follow vs. MGUS: no CRAB, few clonal plasma cells, serum monoclonal protein <3g *AE of Thalidomide = DVTs

Transfusion medicine

Indications for transfusion:  Hb <7 or <10 for acute MI Complications of transfusion: (1) ABO/acute hemolytic transfusion: fever, flank pain, tachycardia, hypotension --> stop transfusion (2) Delayed: elevated bili and LDH, decreased Hb and Haptoglobin, increased retic (3) Post-transfusion purpura: within 1 week, anti-HPA-1a Ab; Tx: IVIg and watch for transfusion-induced thrombocytopenia with next transfusion (4) TRALI (ARDs picture with hypotension after transfusion; Tx: vent/supportive fluids) vs TACO (hypertension; Tx: diuretics)

Other

Sickle Cell: - may present as diffuse pulmonary infiltrates that mimic PE/PNA/appendicitis, but with >2g/dL Hb drop and elevated LDH/retic - acute chest syndrome --> Tx: exchange transfusion if Hb <10 - associated with pulmonary HTN and increased risk for CVA (BUT DO NOT NEED PLAVIX; instead CVA PPx with monthly 2 unit transfusions) - Dx: Hb electrophoresis - chronic Tx with 2 pain crisis/year or h/o ACS: hydroxyurea (but CI in pregnancy and AKI) *vs Fat embolus (long bone fx): fever, CP, thrombocytopenia, multiorgan failure; BAL shows fat bodies *vs. Aplastic crisis (Parvovirus/B19): low retic count

Plasma exchange indications: (1) Guillain Barre (symmetric ascending flaccid paralysis with reduced DTRs) (2) Myasthenia gravis (ocular-->facial -->proximal muscle weakness that doesn’t fatigue) (3) TTP (neurologic symptoms with hemolytic anemia and thrombocytopenia) (4) Goodpasture (anti-GBM nephritis (hematuria, hypertension) with hemoptysis) (5) Cryoglobulinemia (cold-induced nephritis, low complements, associated with HCV) *AE of plasma exchange = hypocalcemia from citrate: perioral numbness, tingling, anxiety/vomit; Tx: calcium gluconate

[cancersfakianakis1]

Blockade of PDGFRβ circumvents resistance to MEK-JAK inhibition via intratumoral CD8 + T-cells infiltration in triple-negative breast cancer

Abstract

Background

Despite the increasing progress in targeted and immune based-directed therapies for other solid organ malignancies, currently there is no targeted therapy available for TNBCs. A number of mechanisms have been reported both in pre-clinical and clinical settings that involve inherent, acquired and adaptive resistance to small molecule inhibitors. Here, we demonstrated a novel resistance mechanism in TNBC cells mediated by PDGFRβ in response to JAK2 inhibition.

Methods

Multiple in vitro (subG1, western blotting, immunofluorescence, RT-PCR, Immunoprecipitation), in vivo and publically available datasets were used.

Results

We showed that TNBC cells exposed to MEK1/2-JAK2 inhibitors exhibit resistant colonies in anchorage-independent growth assays. Moreover, cells treated with various small molecule inhibitors including JAK2 promote PDGFRβ upregulation. Using publically available databases, we showed that patients expressing high PDGFRβ or its ligand PDGFB exhibit poor relapse-free survival upon chemotherapeutic treatment. Mechanistically we found that JAK2 expression controls steady state levels of PDGFRβ. Thus, co-blockade of PDGFRβ with JAK2 and MEK1/2 inhibitors completely eradicated resistant colonies in vitro. We found that triple-combined treatment had a significant impact on CD44+/CD24− stem-cell-like cells. Likewise, we found a significant tumor growth inhibition in vivo through intratumoral CD8+ T cells infiltration in a manner that is reversed by anti-CD8 antibody treatment.

Conclusion

These findings reveal a novel regulatory role of JAK2-mediated PDGFRβ proteolysis and provide an example of a PDGFRβ-mediated resistance mechanism upon specific target inhibition in TNBC.

http://bit.ly/2X4E9dD

[tumimmtxpapers]

Cancer biomarkers for targeted therapy.

Related Articles Cancer biomarkers for targeted therapy. Biomark Res. 2019;7:25 Authors: Liu D Abstract Tumor-associated antigens (TAA) or cancer biomarkers are major targets for cancer therapies. Antibody- based agents targeting the cancer biomarkers include monoclonal antibodies (MoAbs), radiolabeled MoAbs, bispecific T cell engagers, and antibody-drug conjugates. Antibodies targeting CD19, CD20, CD22, CD30, CD33, CD38, CD79B and SLAMF7 are in clinical applications for hematological malignancies. CD123, CLL-1, B cell maturation antigen, and CD138 are targets for cancer immunotherapeutic agents, including the chimeric antigen receptor - engineered T cells. Immune checkpoint inhibitors (ICIs) against PD-1, PD-L1, and CTLA-4 have led to the revolution of cancer immunotherapy. More ICIs targeting IDO, LAG3, TIM-3, TIGIT, SIGLECs, VISTA and CD47 are being explored. Small molecule inhibitors (SMIs) against tyrosine kinase oncoproteins such as BCR-ABL, JAK2, Bruton tyrosine kinase, FLT3, EGFR, ALK, HER2, VEGFR, FGFR, MEK, and MET have fundamentally changed the landscape of cancer therapy. SMIs against BCL-2, IDHs, BRAF, PI3 kinase, mTOR, PARP, and CDKs have become the mainstay in the treatment of a variety of cancer types. To reduce and avoid off-tumor toxicities, cancer-specific TAAs such as CD33 are being manufactured through systems biology approach. Search for novel biomarkers and new designs as well as delivery methods of targeted agents are fueling the next wave of advances in cancer therapy. PMID: 31807308 [PubMed] http://dlvr.it/RKsV6M

[cancersfakianakis1]

JAK/Stat5-mediated subtype-specific lymphocyte antigen 6 complex, locus G6D (LY6G6D) expression drives mismatch repair proficient colorectal cancer

Abstract

Background

Human microsatellite-stable (MSS) colorectal cancers (CRCs) are immunologically “cold” tumour subtypes characterized by reduced immune cytotoxicity. The molecular linkages between immune-resistance and human MSS CRC is not clear.

Methods

We used transcriptome profiling, in silico analysis, immunohistochemistry, western blot, RT-qPCR and immunofluorescence staining to characterize novel CRC immune biomarkers. The effects of selective antagonists were tested by in vitro assays of long term viability and analysis of kinase active forms using anti-phospho antibodies.

Results

We identified the lymphocyte antigen 6 complex, locus G6D (LY6G6D) as significantly overexpressed (around 15-fold) in CRC when compared with its relatively low expression in other human solid tumours. LY6G6D up-regulation was predominant in MSS CRCs characterized by an enrichment of immune suppressive regulatory T-cells and a limited repertoire of PD-1/PD-L1 immune checkpoint receptors. Coexpression of LY6G6D and CD15 increases the risk of metastatic relapse in response to therapy. Both JAK-STAT5 and RAS-MEK-ERK cascades act in concert as key regulators of LY6G6D and Fucosyltransferase 4 (FUT4), which direct CD15-mediated immune-resistance. Momelotinib, an inhibitor of JAK1/JAK2, consistently abrogated the STAT5/LY6G6D axis in vitro, sensitizing MSS cancer cells with an intact JAK-STAT signaling, to efficiently respond to trametinib, a MEK inhibitor used in clinical setting. Notably, colon cancer cells can evade JAK2/JAK1-targeted therapy by a reversible shift of the RAS-MEK-ERK pathway activity, which explains the treatment failure of JAK1/2 inhibitors in refractory CRC.

Conclusions

Combined targeting of STAT5 and MAPK pathways has superior therapeutic effects on immune resistance. In addition, the new identified LY6G6D antigen is a promising molecular target for human MSS CRC.

http://bit.ly/2FQQsEU

[cancersfakianakis1]

Tumor-associated neutrophils induce EMT by IL-17a to promote migration and invasion in gastric cancer cells

Abstract

Purpose

Epithelial to mesenchymal transition (EMT) can contribute to gastric cancer (GC) progression and recurrence following therapy. Tumor-associated neutrophils (TANs) are associated with poor outcomes in a variety of cancers. However, it is not clear whether TANs interact with the EMT process during GC development.

Methods

Immunohistochemistry was performed to examine the distribution and levels of CD66 + neutrophils in samples from 327 patients with GC. CD66b + TANs were isolated either directly from GC cell suspensions or were conditioned from healthy donor peripheral blood polymorphonuclear neutrophils (PMNs) stimulated with tumor tissue culture supernatants (TTCS) and placed into co-culture with MKN45 or MKN74 cells, after which migration, invasion and EMT were measured. Interleukin-17a (IL-17a) was blocked with a polyclonal antibody, and the STAT3 pathway was blocked with the specific inhibitor AG490.

Results

Neutrophils were widely distributed in gastric tissues of patients with GC and were enriched predominantly at the invasion margin. Neutrophil levels at the invasion margin were an independent predictor of poor disease-free survival (DFS) and disease-specific survival (DSS). IL-17a + neutrophils constituted a large portion of IL-17a-producing cells in GC, and IL-17a was produced at the highest levels in co-culture compared with that in TANs not undergoing co-culture. TANs enhanced the migration, invasion and EMT of GC cells through the secretion of IL-17a, which activated the Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT3) pathway in GC cells, while deprivation of IL-17a using a neutralizing antibody or inhibition of the JAK2/STAT3 pathway with AG490 markedly reversed these TAN-induced phenotypes in GC cells induced by TANs.

Conclusions

Neutrophils correlate with tumor stage and predict poor prognosis in GC. TANs produce IL-17a, which promotes EMT of GC cells through JAK2/STAT3 signalling. Blockade of IL-17a signalling with a neutralizing antibody inhibits TAN-stimulated activity in GC cells. Therefore, IL-17a-targeted therapy might be used to treat patients with GC.

http://bit.ly/2RdGd3B

[cancersfakianakis1]

Secretory RAB GTPase 3C modulates IL6-STAT3 pathway to promote colon cancer metastasis and is associated with poor prognosis

Abstract

Background

RAB GTPases are important in the regulation of membrane trafficking and cell movement. Recently, exocytic RABs have received increasing attention in cancer research. However, the functional roles of exocytic RABs in colorectal carcinogenesis remain to be elucidated.

Methods

Immunohistochemistry analysis of a microarray containing 215 colorectal adenocarcinoma tissues was used to identify the association between exocytic RABs and patient prognosis. Complementary functional RAB3C overexpression and knockdown experiments were performed. The molecular mechanism of RAB3C in inducing colon cancer cell metastasis was determined.

Results

High RAB3C expression in patients was found to be significantly associated with advanced pathological stage, distant metastasis and poor prognosis. Multivariate analyses showed that high RAB3C expression was an independent prognostic marker in overall (P = 0.001) and disease-free survival (P < 0.001). Furthermore, our experimental results showed an increase in the migration and invasion ability of RAB3C-overexpressing colon cancer cells and increased metastatic nodules in a mouse metastasis model. The effect of RAB3C-overexpressing cell-conditioned medium was found to significantly promote the migration ability of parental colon cancer cells, thus suggesting that the promotion of migration is exocytosis dependent. Upregulation of other exocytic RABs was also seen in RAB3C-overexpressing cells. Through microarray and proteomics analyses, increased production of multiple cytokines was observed in RAB3C-overexpressing cell lines, and the IL-6 pathway was the top pathway whose members exhibited gene expression changes after RAB3C overexpression, according to Ingenuity Pathway Analysis. Blocking IL-6 with IL-6 antibody treatment or IL-6 knockdown significantly inhibited the migration potential of RAB3C-overexpressing colon cancer cells. In addition, IL-6 was found to induce STAT3 phosphorylation in RAB3C-overexpressing colon cancer cells, thus promoting migration. Ruxolitinib, a JAK2 inhibitor, was found to significantly inhibit RAB3C-induced colon cancer cell migration.

Conclusions

Our study revealed that RAB3C overexpression promotes tumor metastasis and is associated with poor prognosis in colorectal cancer, through modulating the ability of cancer cells to release IL-6 through exocytosis and activate the JAK2-STAT3 signaling pathway. These results further suggest that inhibition of STAT3 phosphorylation in the RAB3C-IL-6-STAT3 axis by using Ruxolitinib may be a new therapeutic strategy to combat metastatic colon cancers.

http://ift.tt/2uiyaH9

[cancersfakianakis1]

Activation of the B-cell receptor successively activates NF-κB and STAT3 in chronic lymphocytic leukemia cells

Abstract

In chronic lymphocytic leukemia (CLL) cells, both interleukin-6 (IL-6) and the B-cell receptor (BCR) activate Janus kinase 2 (JAK2) and induce the phosphorylation of signal transduction and activator of transcription 3 (STAT3) on tyrosine 705 residues. However, whereas IL-6 phosphorylates STAT3 within 15 minutes, stimulation of the BCR with anti–immunoglobulin M (IgM) antibodies phosphorylates STAT3 in 2–4 hours. Here we show that this process takes longer because it requires transcriptional activity of NF-κB. Using an electromobility shift assay, we found that incubation with IgM antibodies for 4 or 18 hours, but not 15 minutes, increased NF-κB DNA-binding of CLL cells and increased binding was translated to increased transcriptional activity. Hence, 42% of the 83 NF-κB target genes were constitutively expressed in all CLL cells prior to any inducible stimuli. However, activation of the BCR increased the number of NF-κB target genes with detectable expression by 23%. Remarkably, prolonged incubation with anti-IgM antibodies induced a time-dependent transcription, production, and secretion of IL-6 protein. The IgM-induced production of IL-6 prompted the phosphorylation of STAT3 on tyrosine residues. This effect was inhibited by the JAK1/2 inhibitor of the JAK/STAT3 pathway ruxolitinib. Taken together, these results suggest that in CLL cells, constitutive tonic activation of NF-κB can be further enhanced by the BCR and that the BCR-induced activation of the JAK/STAT3 pathway depends on the NF-κB–induced production of IL-6. This article is protected by copyright. All rights reserved.

http://ift.tt/2uCeD3J