Integrin alpha 10, CD44, PTEN, cadherin-11 and lactoferrin expressions are potential biomarkers for selecting patients in need of central nervous system prophylaxis in diffuse large B-cell lymphoma

Carcinogenesis, Aug 2017

Lemma, Siria A, Kuusisto, Milla, Haapasaari, Kirsi-Maria, Sormunen, Raija, Lehtinen, Tuula, Klaavuniemi, Tuula, Eray, Mine, Jantunen, Esa, Soini, Ylermi, Vasala, Kaija, et al.

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Integrin alpha 10, CD44, PTEN, cadherin-11 and lactoferrin expressions are potential biomarkers for selecting patients in need of central nervous system prophylaxis in diffuse large B-cell lymphoma

Carcinogenesis Integrin alpha 10, CD44, PTEN, cadherin-11 and lactoferrin expressions are potential biomarkers for selecting patients in need of central nervous system prophylaxis in diffuse large B-cell lymphoma Siria A.Lemma Emails: 1 Milla Kuusisto 1 Kirsi-Maria Haapasaar Raija Sormunen 0 6 Tuula Lehtinen 5 Tuula Klaavuniem 5 Mine Eray 3 Esa Jantunen 8 Ylermi Soini 2 7 Kaija Vasala 4 Jan Böhm Niina Salokorp Petri Koivunen 9 Peeter Karihtal Jussi Vuoristo 0 6 Taina Turpeenniemi-Hujanen 1 Outi Kuittine n 1 0 D,epartment of Pathology, Institute of Diagnostics, Medical Research Center Oulu, Oulu University Hospital , Kajaanintie 50, 90220 Oulu, Finlan 1 D,epartment of Neurosurgery, Medical Research Center Oulu, Oulu University Hospital, University of Oulu , Kajaanintie 50, 90220 Oulu , Finland a 2 Kuopio University Hospital , Puijonlaaksontie 2, 70210 Kuopio, Finlan 3 lDaenpda,rtment of Pathology, FIMLAB, Tampere University Hospital , Teiskontie 35, 33521 Tampere, Finlan 4 nDde,partment of Oncology and Radiotherapy, Central Finland Central Hospital , Keskussairaalantie 19, 40620 Jyväskylä, Fin 5 Department of Oncology, Tampere University Hospital , Teiskontie 35, 33521 Tampere, Finla 6 Bdi,ocenter Oulu, University of Oulu , Kajaanintie 50, 90220 Oulu , Finland 7 dD,epartment of Clinical Pathology and Forensic Medicine, Cancer Center of Eastern Finland, University of Eastern Finland , Puijonlaaksontie 2, 70210 Kuopio , Finland 8 Dde,partment of Medicine, Kuopio University Hospital , Puijonlaaksontie 2, 70210 Kuopio, Finlan 9 nDdepartment of Otorhinolaryngology, Medical Research Center Oulu, Oulu University Hospital, University of Oulu , Kajaanintie 50, 90220 Oulu , Finland - Central nervous system (CNS) relapse is a devastating complication that occurs in about 5% of diffuse large B-cell lymphoma (DLBCL) patients. Currently, there are no predictive biological markers. We wanted to study potential biomarkers of CNS tropism that play a role in adhesion, migration and/or in the regulation of inflammatory responses. The expression levels of ITGA10, CD44, PTEN, cadherin-11, CDH12, N-cadherin, P-cadherin, lactoferrin and E-cadherin were studied with IHC and IEM. GEP was performed to see whether found expressional changes are regulated at DNA/RNA level. IHC included 96 samples of primary CNS lymphoma (PCNSL), secondary CNS lymphoma (sCNSL) and systemic DLBCL (sDLBCL). IEM included two PCNSL, one sCNSL, one sDLBCL and one reactive lymph node samples. GEP was performed on two DLBCL samples, one with and one without CNS relapse. CNS disease was associated with enhanced expression of cytoplasmic and membranous ITGA10 and nuclear PTEN P( < 0.0005, P = 0.002, P = 0.024, respectively). sCNSL presented decreased membranous CD44 and nuclear and cytoplasmic cadherin-11 expressionsP( = 0.001, P = 0.006, P = 0.048, respectively). In PCNSL lactoferrin expression was upregulated P( < 0.0005). IEM results were mainly supportive of the IHC results. In GEP CD44, cadherin-11, lactoferrin and E-cadherin were under-expressed in CNS disease. Our results are in line with previous studies, where gene expressions in extracellular matrix and adhesion-related pathways are altered in CNS lymphoma. This study gives new information on the DLBCL CNS tropism. If further verified, these markers might become useful in predicting CNS relapses. of intercellular adhesion molecule-1 (ICAM-1), this way possibly reducing the recruitment of leukocytes to the inflammatory site ( 23 ). In this work, we performed a screening of the above-me-n tioned molecules with an aim of finding possible biomarkers in order to identify patients with a high risk of CNS recurrence. The expression levels of these molecules were studied by means of immunohistochemistry (IHC) and immunoelectron microscopy (IEM). Gene-expression profiling (GEP) was performed to identify whether the expressional changes seen are regulated at geneexpression level. Materials and methods Patients and samples Introduction Patients included in the study were diagnosed and treated at the University Hospitals of Oulu, Jyväskylä, Kuopio and Tampere between 1997 and 2013. Diffuse large B-cell lymphoma (DLBCL) is the most common The median age of patients at diagnosis was 65 (range 27–82). The co-n lymphoma subtype, representing about 30% of all lymphomas. trol group was selected from patients who did not have any signs of CNS In addition to systemic disease, it can also manifest in the c-en disease during diagnosis, therapy or follow-up from the time of diagnosis tral nervous system (CNS) in the form of secondary CNS invol-ve until the end of the year 2015. Also the control group did not receive CNS ment of systemic DLBCL or as primary CNS lymphoma (PCNSL). prophylaxis. Patient information was collected from the hospital records, kTihnellyamttpehroims aasr(1a)r. e disease, representing 1–2% of all non-Ho-dg ibdnlagitnaGdCewd/anfsorncoom-GllCtehcpteheeldanbforotorymapteop,raaygtiere,enlstausclttwasittaehnddperhuiymsdeadrroaygsesneynastsceerm,yIpPictIeaddnisddeaattshaee., Cinnluci--mnluicdal Of systemic DLBCL patients, around 5% will have disease ber of extranodal lesions. Permission to use patient samples included in involvement in the CNS either during diagnosis, or more co-m the study was granted by the Finnish National Supervisory Authority for monly, a few months afterwards2(,3). The inclusion of ritux-i Welfare and Health (6622/ This study was approved by mab in the treatment has increased the effective control of the Ethics Committee of Oulu University Hospital (42/2010, 23.6.2010). the systemic DLBCL, but the data considering its effect on the The study material for IHC consisted of diagnostic biopsy samples risk of a CNS relapse is contradictory. Before the rituximab era, from 96 patients with newly diagnosed DLBCL, including 15 patients with CNS involvement was usually seen simultaneously with refr-ac PCNSLs, 31 with secondary CNS lymphomas (sCNSLs) and a control group tory, widespread systemic disease 4(,5). This paradigm has of 50 patients with systemic DLBCLs (sDLBCL) that did not receive CNS changed and currently the emergence of solitary CNS relapses fprroomphpylraimxiasr.yThdeiasgCnNoSsiLs.sIaEmMplinesclwudereed dtiwaognPoCsNtiScLlysammpphlenso, doenesasmCpNlSeLs has become an increasing problem. Patients with a CNS relapse (brain biopsy), one sDLBCL and one reactive lymph node sample. GEP have a dismal prognosis. High-dose methotrexate and cyta-ra included two diagnostic lymph node fresh frozen tissue samples, one bine included in the primary treatment of systemic DLBCL seem sDLBCL and one sCNSL sample from primary diagnosis. to reduce the risk of a CNS relapse. However it is associated with considerable toxicity and the identification of patients at high Immunohistochemistry risk of CNS relapse still remains problematic toda6,y7)(. Slide preparation was performed as described earlier except that before Currently the patient selection for CNS prophylaxis is-car deparaffinization the slides were incubated overnight at +37°2C4)(. The ried out by clinical risk assessment based on the following antigen retrieval and the primary antibodies and their concentrations are clinical factors: high International Prognostic Index (IPI) score, presented in Supplementary Table  1, available aCtarcinogenesis Online. advanced stage, elevated lactate dehydrogenase, age > 60 years Sample preparation continued as described in our previous stud9y).(To and involvement of extranodal sites (such as paraspinal or p-ara determine the germinal centre (GC) phenotype of each lymphoma sample, insaasballelotocaitdieonnt,itfyesatepsaotriebnrteapsotp)u.Alattbioenstw,tithhisacl2i0n%icarlisekvaolfuCaNtSionsHtaundsy’ (a9,l2g5o).rithm was used with the same antibodies as in our previous disease, thus the number needed to treat to prevent one relapse Immunoelectron microscopy idsevheilgohp. TChNeSrediisseaaslesoanadsfmaiallltogbroeudpetoefctpeadtibeynttshiwshcolinwi cilall lraistker bTuisfsfuereedpiseacelisnwee(PrBeSf)ixweidthin2.54%% spuacrraofsoer mfoarl6d ehhaydteRTint0h.e1nMimpmheorsspehdaaten-d score. There is an unmet clinical need for biological markers stored in 2.3 M sucrose in PBS. Tissue pieces were placed on specimen that could be used for predicting CNS recurrenc8e).( stubs and frozen in liquid nitrogen. Ultrathin sections (70 nm) were cut Based on our previous study and literature, we hypot-he with a Leica EM UC7 cryo-ultramicrotome. For immunolabelling, ultrathin sized that CXCR4/CXCL12 signalling mediates the CNS homing sections were incubated in 0.1 % glycine + PBS followed by incubation in of lymphoma cells and provokes an epithelial–mesenchymal 1% bovine serum albumin in PBS. All washes and antibody dilutions were transition (EMT)-like process9(). CXCR4/CXCL12 signalling has performed in PBS + 1% bovine serum albumin. After incubation with the been linked to the expression and/or activity of integrins, CD44 primary antibodies the sections were incubated with Protein A-gold c-on (a cell-surface glycoprotein), phosphatase and tensin homolog jugate (10 nm). Rabbit anti-mouse IgG (Jackson ImmunoResearch Europe (PTEN), cadherin-11, N-cadherin and E-cadherin1(0–14). Integrin Lmtedt,hUyKlc)ewllausluosseedaansd aexsaemcoinndeadriynaantTiebcondayi. SSepcirtiitontsrawnesrme iesmsiobnededleedctinron alpha 10 (ITGA10), CD44, PTEN, cadherin-11, cadherin-12 microscope (FEI Company, Eindhoven, the Netherlands) equipped with a (CDH12), N-cadherin and E-cadherin have also been linked to Quemesa digital camera (EMSIS GmbH, Münster, Germany). The antibo-d EMT and associate with the expression of EMT transcriptionf-ac ies used were the same as for IHC. tors (15–21). Lactoferrin levels have been reported to be elevated in the cerebrospinal fluid of patients with CNS involvement of Gene-expression profiling leukemia and lymphoma 2(2). In addition lactoferrin was of The RNA isolation was performed using the RNeasy Mini Kit (Qiagen, USA) interest as it has been shown to downregulate the expression in accordance with the manufacturer’s instructions. A GeneChip protocol was used and the experimental procedures were performed in accordance antibodies used. Nuclear immunostaining was seen with all the with the Affymetrix GeneChip Expression Analysis Technical Manual. Using antibodies used; except for CD44 there was no nuclear immun-o 5 µg of total RNA as template, a double-stranded DNA was synthesized by reactivity. Membranous immunostaining was seen for ITGA10, means of the One-cycle cDNA synthesis kit (Affymetrix, USA) and T7-(dT)24 CD44, PTEN and CDH12. The staining percentages are presented primer. GeneChip Sample Cleanup Module (Qiagen, USA) was used to purify the DNA. By using an in vitro transcription (IVT) labelling kit (Affymetrix, in Supplementary Table  2, available aCtarcinogenesis Online. USA) according to the manufacturer’s instructions, the IVT was performed Figure 1 presents IHC staining patterns. to produce biotin labelled complementary RNA. Biotinylated complem-en tary RNA was cleaned using the GeneChip Sample Cleanup Module (Qiagen), Immunoelectron microscpy expression in fragmented to 35–200 nucleotides, and hybridized to Affymetrix Human lymphoma samples Genome U133 Plus 2.0 arrays that contain ~50500 human transcripts. After In PCNSL samples, the strongest expressions were seen for washing, the array was stained with streptavidin–phycoerythrin (Molecular CD44, PTEN, cadherin-11 and N-cadherin. ITGA10 membranous Probes). The staining signal was amplified by biotinylated anti-streptavidin expression was substantial in sCNSL. CD44 membranous and (Vector Laboratories) followed by second staining with streptavidin-ph-yco erythrin, and then scanned on the GeneChip Scanner 3000. CDH12 nuclear expressions were substantial in sDLBCL. Strong nuclear expression of CDH12 was also detected in lymph node Analysis hyperplasia. In the analysis of IHC staining, the location of positivity in the m-alig The proteins studied were also detected outside of ly-m nant B-cells was determined separately in the nucleus, cytoplasm and/ phoma cells. Particularly lactoferrin was seen outside of -lym or cell membrane. Then the percentage of positive cells was estimated phoma cells in all samples, most strongly in PCNSL. In sDLBCL separately for each location, from 0 to 100% with 5% accuracy. Micrographs and lymph node hyperplasia CDH12 was similarly expressed from IHC staining patterns were obtained using an Olympus DP11 digital in normal lymphocytes as in lymphoma cells. CDH12 staining microscope camera (Olympus, Center Valley) and an Olympus BX41 mic-ro was also seen outside of lymphoma cells in PCNSL and sCNSL scope. To import micrographs an HP Photo and Imaging software package samples. In PCNSL, CD44 seemed to be expressed on the plasma (Hewlett-Packard Company, Palo Alto) was used. membranes of the cells that came into contact with lymphoma scopTeh.eAI EgMensearmalpalsessewssemreenretvwiewasedmuasdiengofatthreanexspmriessssiioonneplaectttreornn,mtaick-rinog cells with cell protrusions. PTEN was similarly expressed in into account the location (nucleus, cytoplasm and/or cell membrane) and PCNSL and sCNSL samples in cells that had long cell protrusions the amount of positivity detected. Micrographs of each sample were taken that came into contact with lymphoma cells. It was expressed in using a Quemesa digital camera (EMSIS GmbH, Münster, Germany) and a the membranes of these cells. The precise cell type could not be Tecnai Spirit transmission electron microscope (FEI Company, Eindhoven, determined. Possibly these were antigen-presenting microglial the Netherlands) as part of the analysis. cells. In PCNSL samples ITGA10 was also detected in cells that The expression data gained from GEP was analysed using dChip so-ft were not lymphoma cells. In addition it was seen in myelinated ware ( 26 ). and non-myelinated neurons. Low expression of N-cadherin For statistical analysis, IBM SPSS Statistics for Windows 20.0 software was detected in PCNSL in cells surrounding lymphoma cells. (IBM Corp. Released 2011. Armonk, NY) was used. In order to analyse the In sCNSL, the expression in surrounding cells was higher. High associations between diagnosis (sDLBCL versus sCNSL) and the IHC pr-o tein expressions, the cut-off points for the latter were chosen using the expression of cadherin-11 was seen in the plasma membranes receiver operating characteristic curve. The area under the curve (AUC) of cells surrounding the lymphoma cells in the sCNSL sample. shows sensitivity versus 1-specificity. Only values greater than 0.5 were These cells could possibly represent antigen-presenting mic-ro taken into account, values closer to 1.0 possessing a better discriminatory glial cells. Supplementary Figure  1, available Caatrcinogenesis power. Due to the small study material, AUCs were relatively low. Cut- Online, presents electronmicroscopic expression in these cells. off points were chosen by estimating a good balance between sensitivity Supplementary Table  3, available aCtarcinogenesis Online, and specificity. AUCs and cut-off points for proteins with statisticall-y sig represents immunoelectron microscopy expression in all sa-m nificant associations with diagnosis are presented in the results section. ples. Figure 2 presents electronmicroscopic expression. Receiver operating characteristic curve-based cut-off points could not be chosen for all the proteins, due to the lack of discriminatory power. For Differences in gene-expression the analysis of the associations between protein expressions and clinical parameters, median values were used as cut-off points for all the proteins, The molecules studied were found in GEP data but only CD44, with these varying from 0.00 to 100.00. cadherin-11, lactoferrin and E-cadherin presented differences IPI values were divided into two groups, with group one including in the gene expression levels, being underexpressed in sCNSL patients with IPI points from 0 to 2, and group two including IPI points from compared to sDLBCL T(able 1). 3 to 5. The number of extranodal lesions was also divided into two groups, with group one including patients with zero or one extranodal lesions, and Associations with central nervous system group two including patients with two or more extranodal lesions. A Kruskal–Wallis test for continuous variables was performed inc-lud involvement ing all three different diagnoses (sDLBCL, sCNSL and PCNSL) and a Mann– There were statistically significant differences between the WhitneyU test for two diagnostic groups (sCNSL and PCNSL). A chi-square expression of ITGA10, CD44, PTEN, cadherin-11 and lactoferrin test was used to determine the associations between protein expressions stainings in different diagnostic groups. The differences gained and clinical parameters as well as protein expressions and diagnosis from the Kruskal–Wallis test for continuous variables with all (sDLBCL and sCNSL). three diagnoses are presented here, mean and median values representing the staining  2 represents the Results differences gained from the Mann–WhitneyU test comparing two diagnoses; sCNSL versus PCNSL andTable  3 the results of Immunohistochemical expression in lymphoma the chi-square test with chosen cut-off pointTsa(bles 2 and 3). samples ITGA10 cytoplasmic expression was lowest in sDLBCL (mean E-cadherin did not show any immunoreactivity. For ITGA10, rank = 22.62, median = 0.00), higher in sCNSL (mean rank = 33.20, CD44, PTEN, cadherin-11, N-cadherin, P-cadherin and lact-o median  =  50.00) and highest in PCNSL (mean rank  =  46.85, ferrin, cytoplasmic immunostaining was seen with all the median = 100.00) (P < 0.0005) (Figure 3A). ITGA 10 membranous expression was lowest in sDLBCL (mean rank  =  25.40, median 0.00), higher in PCNSL (mean rank  =  26.08, median  =  0.00) and highest in sCNSL (mean rank = 40.25, median = 100) (P = 0.002) (Figure 3B). CD44 membranous expression was lowest in sCNSL (mean rank  =  34.82, median  =  50.00), higher in sDLBCL (mean rank  =  46.22, median  =  70.00) and highest in PCNSL (mean rank = 67.54, median = 100.00) (P = 0.001) (Figure 3D). PTEN nuclear expression was lowest in sDLBCL (mean rank = 25.72, median = 0.00), higher in PCNSL (mean rank = 36.04, median  =  0.00) and highest in sCNSL (mean rank  =  38.37, median = 5.00) (P = 0.024) (Figure 3C). Cadherin-11 nuclear expression was lowest in sCNSL (mean rank  =  34.02, median  =  30.00), higher in sDLBCL (mean rank  =  51.80, median  =  100.00) and highest in PCNSL (mean rank = 52.23, median = 100.00) (P = 0.006) (Figure 3E). Cadherin-11 cytoplasmic expression was lowest in sCNSL (mean rank = 41.52, median 0.00), higher in sDLBCL (mean rank 46.64, median = 0.00) and highest in PCNSL (mean rank = 54.00, median 0.00) (P  =  0.048). When comparing the expression between just two groups, sCNSL and sDLBCL, the result was non-significant. Lactoferrin cytoplasmic expression was lowest in sDLBCL (mean rank  =  28.50, median  =  0.00), higher in sCNSL (mean rank  =  30.18, median  =  0.00) and highest in PCNSL (mean rank  =  52.85, median  =  100.00) (P  <  0.0005) (Figure  3F). When comparing the expression between just two groups, sCNSL and sDLBCL, the result was non-significant. Associations with clinical parameters CDH12 nuclear and ITGA10 membranous expressions had a positive association with the number of extranodal lesions Table 1. Gene-expression profiling results with CNS tropism, while CD44 and cadherin-11 expressions seemed to be protective of secondary CNS disease. Interestingly Protein Fold-change though, CD44, cadherin-11 and lactoferrin expressions were ITGA10 NC highest in PCNSL samples. CD44 –3.93 CNS recurrence occurs in about 5% of systemic DLBCL patients PTEN NC (2,3). It is a difficult complication with a very poor prognosis. Cadherin-11 –42.04 Abramson et  al. conducted a retrospective study with results CDH12 NC that were later confirmed in a prospective CRY study by Nordic N-cadherin NC Lymphoma Group, showing that CNS relapse risk can be reduced P-cadherin NC with high-dose methotrexate containing prophylactic che-mo Lactoferrin –1200.21 therapy ( 6,27 ). This conclusion was further supported by a ret-ro E-cadherin –16.90 spective study by Ferreriet  al ( 28 ). However, because high-dose methotrexate is a resource-demanding therapy with conside-ra Negative fold-changes mark decreased expression in sCNSL compared to ble toxicity, it should be limited to patients with a high risk of CNS sDLBCL. The fold change for CD44 is a mean of five different values from five recurrence. Currently identification of these high-risk patients is different probes. NC, no change. performed based on clinical factors, which unfortunately have a poor specificity 8(). Thus there is a need to define biological mar-k (P = 0.026 and P = 0.006, respectively). CD44 membranous expre-s ers with a higher specificity for separating patients in need of sion had an inverse association with the number of extranodal CNS prophylactic treatment. Currently there are suggestions that lesions (P = 0.037). CD12 nuclear, lactoferrin cytoplasmic, ITGA10 double-hit genotype, non-GC phenotype, as well as CD5 positivity cytoplasmic and ITGA membranous expressions had a positive may have some prognostic relevance in this matte2r9(–31). association with the IPI scoreP( = 0.010, P = 0.029, P = 0.022 and Understanding the pathophysiology of CNS lymphomas P  =  0.007, respectively). PTEN nuclear expression had a po-si has been a challenge for years. An attractive hypothesis is that tive association with lactate dehydrogenasPe  =( 0.002). Higher these malignant cells originate from extracranial sites but end CD44 membranous expression was associated with the non-GC up homing into the CNS due to highly selective CNS tropism. phenotype (P = 0.049). CD44 membranous expression also had a Results from recent studies have supported this hypothesi3s2(). positive association with age over 60P( = 0.034). In studies with GEP, differences in transcription of several p-ro teins have been observed when comparing normal lymphatic tissue and nodal or extranodal DLBCL with PCNSL. Our results Discussion are in line with these previous findings because it seems that In this work, we studied the biology of DLBCL’s CNS tropism with adhesion-, migration- and ECM-related molecules play a pivotal IHC, IEM and GEP with the aim of finding biological markers that role in CNS involvement of DLBCL3( 3,34 ). would define DLBCL patients at high risk of CNS recurrence. We In our previous study of CNS lymphomas, we assumed that found that elevated levels of ITGA10 and PTEN were associated CXCR4/CXCL12 and CXCR5/CXCL13 axis play an important role CD44, M Lactoferrin, C P cadherin, N ITGA10, C ITGA10, M Protein, location CD44, M PTEN, N ITGA10, N ITGA10, C ITGA10, M Cadherin-11, N Median PCNSL 100 100 20 100 0 Association ↓ ↑ ↓ ↑ ↑ ↓ P value in CNS tropism of DLBCL 9(). Concordantly Rubensteinet al. ( 35 ) the CNS tropism in this way. In our study nuclear PTEN was the have shown that CXCL12 and CXCL13 act as mediators of CNS only protein associated with increased lactate dehydrogenase lymphoma cell chemotaxis. In line with these findings, CXCR4/ levels. CXCL12 signalling has been shown to induce EMT or an EMT-like Cadherin-11 cytoplasmic and nuclear expressions were process that enables cell migration towards the expression of highest in PCNSL, yet the latter was associated inversely with chemokine ligands and invasive abilities3( 6–39 ). CXCR4/CXCL12 sCNSL. In contrast to this, in epithelial cancers, cadherin-11 is signalling has also been shown to induce integrin and CD44 often associated with mesenchymal features gained through activation and also CD44 localization to the leading edge during EMT (17). migration in human CD34+ stem/progenitor cells. These events In our results, lactoferrin expression was highest in PCNSL together with integrin expression facilitate cell attachment toand second highest in sCNSL when studied with IHC and IEM. the extracellular space as well as the invasion and mobilization When comparing sDLBCL and sCNSL, no statistically significant of cells 1(0,12). CD44 affects human haematopoietic progen-i differences were seen. In GEP, higher expression level was seen tor cell trafficking to the BM via interactions with hyaluronicin sDLBCL samples but GEP did not include PCNSL samples that acid (10). It is noteworthy that hyaluronic acid is also the main had the most abundant expression in IHC and IEM. Lactoferrin component of brain extracellular matr4ix0)(. He et al. (41) found has been shown to be produced in the CNS by activated micr-o that perivascular tumour cells in PCNSL showed a strong CD44 glial cells and its expression has been associated with neu-ro expression explaining the perivascular staining pattern. Our logical disorders affecting the CNS, such as Alzheimer’s and results are in line with this data describing the highest CD44 Parkinson’s diseases. It seems that lactoferrin expression is expression in PCNSLs. upregulated as a consequence of inflammatory conditions and Our findings of integrin expression were in line with the under oxidative stress 4(8). Most likely this could also be the hypothesis of cell migration, as ITGA10 showed a strong CNS case in PCNSL. tropism favouring function4(2,43). The expression of membra- Taken together, our findings support the hypothesis that nous ITGA10 had a positive association with two of the known CXCR4 signalling might participate in the initiation of l-ym clinical risk factors for CNS tropism: the number of extranodal phoma cells homing into the CNS. This again may enable the lesions and higher IPI score. morphological and expressional changes seen in this study. In a PTEN is generally regarded as a tumour suppressor gene and work by Ricciardiet al. (49) mesenchymal stromal cell-like f-ea in cancers its loss is associated with increased proliferation rate tures gained through EMT conferred cancer cells with immune(44). Loss of PTEN expression has also been shown to occur in modulatory properties which again possibly attenuate cancer some B-cell lymphomas 4(5,46). We found its nuclear location cell immune escape and progression. to be associated with enhanced CNS tropism. In a work by Goh When the IHC, IEM and GEP results were compared they et al. nuclear trafficking of PTEN after an injury to the neurons were mostly in concordance with each other. All of the m-ole was shown to lead to survival and thus to protect these cells cules studied were found in GEP and four presented with genefrom cell death, probably due to the depletion of PTEN from its expressional changes. The expressional changes were similar functional place, the cytoplasm and cell membran47e)(. Loss of to IHC and IEM results, except that E-cadherin was not seen PTEN has been shown to result in increased CXCR4 and CXCL12 to be expressed at a protein level. Lactoferrin expression also expression and invasion in prostate cancer cell1s1(). If nuclear presented with minor differences. The small number of GEP localization is able to inhibit the cell cycle regulatory functionssamples used in this study limits the reliability of the results. of PTEN, it could be that it can also lead to increased CXCR4 However, concordant results seen in larger IHC and IEM mat-eri expression as seen when PTEN expression is lost, and enhance als indicate that these findings seem to present a relevant event in DLBCL CNS tropism. The few discrepancies may be explained by the methods used; IHC measures the expression of the stu-d ied proteins within a sample tissue whereas GEP measures the expression of specific messenger RNAs in the sample. IHC and IEM results were also mostly in concordance with each other, with only a couple of differences. IHC results represent a collection of multiple samples and a large number of cells are Biocenter Oulu EM laboratory for the preparation of IEM evaluated. IEM expression on the other hand represents one or samples. two samples, and from those a single cell per view. This might Conflict of Interest Statement: None declared. cause a selection bias if the expression in this specific sample is below average. Using light microscopy, it is harder to tell c-yto References plasmic and membranous staining apart than it is in electron microscopy. This might represent another bias to the results. 1. Hochberg, F.H. et  al. (2007) Primary CNS lymphoma. Nat. Clin. Pract. Our findings imply that some biological events are co-m 2. BNoesu,rGo.lM., .3,e2t4 a–l3.5(.1998) For which patients with aggressive non-Ho-dg mon in both primary and secondary CNS lymphomas. Yet the kin’s lymphoma is prophylaxis for central nervous system disease fact that PCNSL samples are brain biopsies and sCNSL sa-m mandatory? Dutch HOVON Group. Ann. Oncol., 9, 191–194. ples from lymph nodes from a time of the primary diagnosis, 3. van Besien, K. et al. (1998) Risk factors, treatment, and outcome of c-en the differences in the microenvironment might also lead to the tral nervous system recurrence in adults with intermediate-grade and seemingly different results observed between these two groups, immunoblastic lymphoma. Blood, 91, 1178–1184. and this impact could not be excluded in this study. In addition, 4. Tai, W.M. et al. (2011) Central nervous system (CNS) relapse in diffuse PCNSL and sCNSL are two different entities and there might also large B cell lymphoma (DLBCL): pre- and post-rituximab. Ann. Hem-a be differences due to this. tol., 90, 809–818. In this work, we have shown that lymphoma CNS tropism 5. Yamamoto, W. et al. 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(2003) cell migration and invasion, and with the current GEP data of Intensive conventional chemotherapy (ACVBP regimen) compared CNS lymphomas where differences in the expression of adh-e with standard CHOP for poor-prognosis aggressive non-Hodgkin ly-m sion and ECM-related genes have been seen 3( 3,34 ). These EMT- phoma. Blood, 102, 4284–4289. like changes seen in our study might also attenuate lymphoma 8. Siegal, T. et al. (2012) CNS prophylaxis in diffuse large B-cell lymphoma: cell immune escape (49). To our knowledge, this is the first 9. iLfe,mwhmean,,Sh.oAw.eatn iBllaorocdhReemv.o,k2i6n, e97r–e1c0e6p.tor profiles in ly-m study exploring the role of ITGA10, CD44, PTEN, cadherin-11, phomas with central nervous system involvement - possible bioma-rk CDH12, N-cadherin, P-cadherin, lactoferrin and E-cadherin in ers for patient selection for central nervous system prophylaxis, a DLBCLs and especially in CNS lymphomas. The present study retrospective study. Eur. J. Haematol., 96, 492–501. was a retrospective one and the size was too small to calculate 10. Avigdor, A. et al. (2004) CD44 and hyaluronic acid cooperate with SDF-1 the positive and negative prognostic values of these markers as in the trafficking of human CD34+ stem/progenitor cells to bone m-ar well as to perform multivariate analyses comparing indepe-nd row. Blood, 103, 2981–2989. ent values of these markers with clinical factors. Currently the11. Conley-LaComb, M.K. et al. (2013) PTEN loss mediated Akt activation most powerful markers for prediction of CNS tropism seem to promotes prostate tumor growth and metastasis via CXCL12/CXCR4 be non-GC phenotype, double-hit genotype and CD5 positi-v signaling. Mol. Cancer, 12, 85. ity ( 28–31 ). From our candidate markers, only CD44 expre-s 12. LPeFAle-d1,, VAL.Ae-t4 ,aal.n(d20V0L0A) -T5hoenchimemmoaktiunree ShDuFm-1anacCtDiv3a4t(e+s) ctehlelsi:nrtoelgeriinns sion was associated with non-GC phenotype, implying that the transendothelial/stromal migration and engraftment of NOD/SCID rest of the proteins studied may give new prognostic value for mice. Blood, 95, 3289–3296. CNS relapse prediction in both GC and non-GC phenotypes. 13. Sobolik,T. et al. (2014) CXCR4 drives the metastatic phenotype in breast Unfortunately translocation data was not available from our cancer through induction of CXCR2 and activation of MEK and PI3K cases. However the findings are able to support a hypothesis pathways. Mol. Biol. Cell, 25, 566–582. that cytoplasmic and membranous ITGA10, nuclear PTEN and 14. Zhu, Y. et al. (2013) The effect of CXCR4 silencing on epithelial-mese-n membranous CD44 expressions should be studied as promi-s chymal transition related genes in glioma U87 cells. Anat. Rec. (H-obo ing candidates to be used as biological markers for screening ken)., 296, 1850–1856. of systemic DLBCL patients at elevated CNS relapse risk. These 15. cMear,cJ.eeltl saal.n(d20i1n6c)rCeaasdehseprrino-g1r2eesnsihoannbcyesprpormoloifteirnagtEioMnTi.nTucmoloourrecBtiaoll.-c,3a7n, results are highly preliminary and need to be validated in a 9077–9088. larger prospective trial. 16. Nieto, M.A. (2011) The ins and outs of the epithelial to mesench-y mal transition in health and disease. Annu. Rev. Cell Dev. Biol., 27, Supplementary material 347–376. Supplementary material is available Caatrcinogenesis online. 17. uNparmeg,uEl.aHt.inegt caald.h(2er0i1n4-)11ZEaBn2d-Sinp1tegcroioαnp5eerxaptrioenssiionnd.uCcaersciinnovgaesinoensisb,y 35, 302–314. Funding 18. Song, M.S. et  al. (2012) The functions and regulation of the PTEN This study was supported by grants from the Cancer Society tumour suppressor. Nat. Rev. Mol. Cell Biol., 13, 283–296. of Northern Finland, Orion-Farmos Research Foundation, the 19. sViise.irMao, lA..CF.aentc aerl,. 1(240, 1157)8P.-cadherin and the journey to cancer metas-ta Finnish Medical Society Duodecim, Finnish Cultural Foundation 20. Xu, H. et al. (2015) The role of CD44 in epithelial-mesenchymal tra-nsi and the Väisänen Fund in Terttu Foundation. tion and cancer development. Onco. Targets. Ther., 8, 3783–3792. 21. Mochmann, L.H. et al. (2014) ERG induces a mesenchymal-like state as-so Acknowledgements ciated with chemoresistance in leukemia cells. Oncotarget, 5, 351–362. 22. Oberg, G. et al. 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Lemma, Siria A, Kuusisto, Milla, Haapasaari, Kirsi-Maria, Sormunen, Raija, Lehtinen, Tuula, Klaavuniemi, Tuula, Eray, Mine, Jantunen, Esa, Soini, Ylermi, Vasala, Kaija, Böhm, Jan, Salokorpi, Niina, Koivunen, Petri, Karihtala, Peeter, Vuoristo, Jussi, Turpeenniemi-Hujanen, Taina, Kuittinen, Outi. Integrin alpha 10, CD44, PTEN, cadherin-11 and lactoferrin expressions are potential biomarkers for selecting patients in need of central nervous system prophylaxis in diffuse large B-cell lymphoma, Carcinogenesis, 2017, 812-820, DOI: 10.1093/carcin/bgx061