EZH2 mutations at diagnosis in follicular lymphoma: a promising biomarker to guide frontline treatment.

EZH2 is mutated in nearly 25% of follicular lymphoma (FL) cases. Little is known about how EZH2 affects patients' response to therapy. In this context, the aim of this study was to retrospectively analyze the frequency of mutations in EZH2 at diagnosis in tissue and ctDNA in patients with FL and to assess the patients' outcomes after receiving immunochemotherapy, depending on the EZH2 mutation status. Among the 154 patients included in the study, 27% had mutated EZH2 (46% with high-grade and 26% with low-grade FL). Of the mutated tissue samples, the mutation in ctDNA was identified in 44% of cases. EZH2 mutation in ctDNA was not identified in any patient unmutated in the tissue.Unmutated patients who received R-CHOP had significantly more relapses than patients who received R-Bendamustine (16/49 vs. 2/23, p = 0.040). Furthermore, our results show that patients with mutated EZH2 treated with R-CHOP vs. those treated with R-Bendamustine present a lower incidence of relapse (10% vs. 42% p = 0.09 at 4 years), a higher PFS (92% vs. 40% p = 0.039 at 4 years), and higher OS (100% vs. 78% p = 0.039 at 4 years). Based on these data, RCHOP could be a more suitable regimen for mutated patients, and R-bendamustine for unmutated patients. These findings could mean the first-time identification of a useful biomarker to guide upfront therapy in FL.

Haplo-Cord transplantation compared to haploidentical transplantation with post-transplant cyclophosphamide in patients with AML.

For patients with AML, the best alternative donor remains to be defined. We analyze outcomes of patients who underwent myeloablative umbilical cord blood or haploidentical hemopoietic stem cell transplantation (HSCT) in Spain. Fifty-one patients underwent single umbilical cord blood transplantation supported by a third party donor (Haplo-Cord) between 1999 and 2012, and 36 patients received an haploidentical HSCT with post-transplant cyclophosphamide (PTCY-haplo) between 2012 and 2014 in GETH centers. The Haplo-Cord cohort included a higher proportion of patients with high disease risk index and use of TBI in the conditioning regimen, and hematopoietic cell transplantation-age Comorbidity Age Index was higher in PTCY-haplo patients. Cumulative incidence of neutrophil engraftment was 97% in the Haplo-Cord and 100% in the PTCY-haplo group, achieved in a median of 12 and 17 days, respectively (P=0.01). Grade II-IV acute GvHD rate was significantly higher in the PTCY-haplo group (9.8% vs 29%, P=0.02) as well as chronic GvHD rates (20% vs 38%, P=0.03). With a median follow-up of 61 months for the Haplo-Cord group and 26 months for the PTCY-haplo cohort, overall survival at 2 years was 55% and 59% (P=0.66), event-free survival was 45% vs 56% (P=0.46), relapse rate was 27% vs 21% (P=0.79), and non-relapse mortality was 17% vs 23% (P=0.54), respectively. In this multicenter experience, Haplo-Cord and PTCY-haplo HSCT offer valid alternatives for patients with AML. Neutrophil engraftment was faster in the Haplo-Cord cohort, with similar survival rates, with higher GvHD rates after haploidentical HSCT.

Reliable quantification of hematopoietic chimerism after allogeneic transplantation for acute leukemia using amplification by real-time PCR of null alleles and insertion/deletion polymorphisms.

Increasing mixed chimerism (MC) after allogeneic stem cell transplantation (SCT) has been associated with a high risk of relapse in acute leukemia. We evaluated a new method for chimerism detection, based on the quantitative real-time PCR (qrt-PCR) amplification of null alleles or insertion/deletion polymorphisms (indels). All qrt-PCR assays with null alleles and indels attained a sensitivity of at least 10(-4), as well as good intra- and interassay concordance, and a high accuracy in experiments with cell mixtures. Informativeness was found in 80.3% of the 61 donor/recipient pairs tested. Nonrelapsed patients showed a progressive decrease in peripheral blood chimerism to values below 0.01% (complete chimerism (CC)). Bone marrow chimerism failed to reach CC more than 4 years after SCT. Increasing MC was observed prior to relapse in 88.2% of patients. Compared with conventional PCR amplification of variable number of tandem repeats, qrt-PCR predicted a significantly higher number of relapses (88.2 vs 44.4%) with a median anticipation period of 58 days. In conclusion, chimerism determination by qrt-PCR amplification of null alleles and indels constitutes a useful tool for the follow-up of patients with acute leukemia after SCT, showing better results than those obtained with conventional PCR.

UGT2B17 minor histocompatibility mismatch and clinical outcome after HLA-identical sibling donor stem cell transplantation.

Minor histocompatibility Ags (mHags) have been implicated in the pathogenesis of GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Uridine diphospho-glucuronosyltransferase 2B17 (UGT2B17) gene deletion may act as a mHag and its association with acute GVHD (aGVHD) has been described. We retrospectively studied the clinical impact of a UGT2B17 mismatch in a cohort of 1127 patients receiving a HSCT from an HLA-identical sibling donor. UGT2B17 mismatch was present in 69 cases (6.1%). Incidence of severe aGVHD was higher in the UGT2B17 mismatched pairs (22.7% vs 14.6%), but this difference was not statistically significant (P: 0.098). We did not detect differences in chronic GVHD, overall survival, relapse-free survival, transplant-related mortality or relapse. Nevertheless, when we analyzed only those patients receiving grafts from a male donor (616 cases), aGVHD was significantly higher in the UGT2B17 mismatched group (25.1% vs 12.8%; P: 0.005) and this association was confirmed by the multivariate analysis (P: 0.043; hazard ratio: 2.16, 95% confidence interval: 1.03-4.57). Overall survival was worse for patients mismatched for UGT2B17 (P: 0.005). We conclude that UGT2B17 mismatch has a negative clinical impact in allogeneic HSCT from HLA-identical sibling donors only when a male donor is used. These results should be confirmed by other studies.

Busulfan-based reduced intensity conditioning regimens for haploidentical transplantation in relapsed/refractory Hodgkin lymphoma: Spanish multicenter experience.

Relapsed or refractory Hodgkin lymphoma (advanced HL) still remains a therapeutic challenge. Recently, unmanipulated haploidentical related donor transplant with reduced conditioning regimen (HAPLO-RIC) and post-transplant cyclophosphamide (PT-Cy) as GvHD prophylaxis has became a promising rescue strategy potentially available to almost every patient. This paper reports our multicenter experience using an IV busulfan-based HAPLO-RIC regimen and PT-Cy in the treatment of 43 patients with advanced HL. Engraftment occurred in 42 patients (97.5%), with a median time to neutrophil and platelet recovery of 18 and 26 days. Cumulative incidences of grades II-IV acute GvHD and chronic GvHD were 39% and 19%, respectively. With a median follow-up of 25.5 months for survivors, 27 patients are alive, with 22 of them disease free. Cumulative incidences of 1-year non-relapse mortality and relapse at 2 years were 21% and 24%, respectively. The estimated 2-year event-free survival (EFS) and overall survival (OS) were 48% and 58%, respectively. CR prior to HAPLO-RIC correlated with better EFS (78.5% vs 33.5%; P=0.015) and OS (86% vs 46%; P=0.044). Our findings further confirm prior reports using HAPLO-RIC in advanced HL in a multicenter approach employing an IV busulfan-based conditioning regimen.

In vivo adhesion of malignant B cells to bone marrow microvasculature is regulated by α4ß1 cytoplasmic-binding proteins.

Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4ß1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are ß1-integrin-binding partners that regulate ß1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4ß1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4ß1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4ß1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4ß1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4ß1 activity.

Lymphoma associated chromosomal abnormalities can easily be detected by FISH on tissue imprints. An underused diagnostic alternative.

BACKGROUND: Fluorescence in situ hybridisation (FISH) is useful for detecting specific chromosomal abnormalities in various tumours. In lymphomas, diagnosis is frequently made using paraffin wax embedded tissue. However, FISH performed under these conditions presents potential technical problems and difficulties in interpretation. AIMS: To show that FISH using tissue imprints and cytopreps or alternatively, bone marrow (BM) smears, constitutes an easy and rapid strategy to overcome these constraints. METHODS: The study comprised 46 patients with lymphoma. Sixty nine tissue imprints, cytopreps, or BM smears were analysed by FISH. Dual colour, dual fusion FISH probes were used to detect the t(8;14), t(11;14), and t(14;18) translocations, whereas a dual colour breakapart FISH probe was used to detect chromosomal translocations involving the BCL6 gene. RESULTS: Tissue imprints and cytopreps were successfully hybridised in all 52 cases, whereas hybridisation was successful in 16 of 17 archival BM smears. All patients could be analysed to identify either the presence or absence of chromosomal translocations. CONCLUSIONS: The use of tissue imprints, cytopreps, or BM smears to identify chromosomal abnormalities by FISH is a rapid and useful ancillary approach for diagnostic purposes. Therefore, it could be used on a routine basis whenever fresh samples are available.

Improving chimaerism quantification in bone marrow transplant recipients by image processing and analysis after restriction endonuclease in situ digestion (IPA-REISD).

Restriction endonuclease in situ digestion (REISD) with Sau3A of human metaphase chromosomes and interphase nuclei produces a conspicuous banding pattern involving pericentromeric regions of chromosomes 9 and 3. Constitutive heterochromatin of chromosome 9 is never digested by this enzyme while that of chromosome 3 is polymorphic, giving rise to three possible karyotypes: homozygous digested (3--), homozygous undigested (3++) or heterozygous individuals (3+-). Discrimination of this polymorphism between donor and recipient cells constitutes a rapid sex-independent method to monitor quantitatively the chimaerism achieved after bone marrow transplantation. An image processing and analysis (IPA)-assisted procedure which resolves residual fluorescent regions in metaphase chromosomes or interphase nuclei after REISD has been developed. IPA-REISD has interesting advantages over the basic REISD method by allowing a rapid, objective and precise discrimination of the polymorphism in large cell samples.

Quantification of donor and recipient hemopoietic cells by real-time PCR of single nucleotide polymorphisms.

Analysis of changes in recipient and donor hemopoietic cell origin is extremely useful to monitor the effect of stem cell transplantation (SCT) and sequential adoptive immunotherapy by donor lymphocyte infusions (DLI). We developed a sensitive and accurate method to quantify the percentage of recipient and donor cells by real-time PCR using single nucleotide polymorphisms (SNPs) as markers. Allele-specific PCR of seven SNPs resulted in specific markers for donor or recipient in 97% of HLA-identical sibling pairs. Both, recipient- and donor-derived hemopoietic cells can be simultaneously analyzed in 67% sibling pairs. We expect this can be increased to approximately 99% by developing three additional SNP-PCR. Serial dilution of SNP-positive DNA into either SNP-negative DNA or water revealed a detection limit of 0.1-0.01% depending on the amount of input DNA and start C(t) of the used SNP-PCR. Application of our real-time SNP-PCR method for a CML patient treated by allogeneic SCT and DLI demonstrated its feasibility to follow donor T-cell chimerism and early detection of residual and recurrent autologous hemopoiesis in response to treatment. This detailed monitoring of the genetic origin of hemopoietic cells, in particular immune effector cells and target cells after SCT and DLI, may substantially contribute to understanding of the mechanisms that play a role in the success of treatment.

Restriction endonuclease in situ digestion (REISD): a novel quantitative sex-independent method to analyze chimerism after bone marrow transplantation.

Restriction endonuclease (RE) in situ digestion (REISD) of human metaphase chromosomes and interphase nuclei may uncover cryptic polymorphisms. This technique can be applied to identify the individual origin of cells and thus analyze the hemopoietic chimerism that eventually results in leukemic patients after allogeneic bone marrow transplantation (BMT). In the current study, results of REISD with different REs are shown. In particular, the use of Sau 3A reveals a polymorphism for constitutive heterochromatin of chromosome 3 and may differentiate BMT donor (D) and recipient (R) cells. Once pre-BMT characterization shows a different Sau 3A digestion pattern of D and R cells, it is possible to monitor the development of hematopoietic cell populations in the R bone marrow after BMT. A panel of 24 patients who underwent BMT and their Ds were analyzed. The method presented here allowed cells from D and R to be distinguished, and therefore to quantify the post-BMT hemopoletic chimerism, in 6 (25%) of the cases. This quantitative and sex-independent genetic approach to the study of hemopoietic chimerism has already shown itself to be useful in patients with leukemia who require a BMT, but could also be extended to other transplant situations.

[Hospital biomedical research through the satisfaction of a Health Research Institute professionals]. / La investigación biomédica hospitalaria a través de la satisfacción de los profesionales de un Instituto de Investigación Sanitaria.

OBJECTIVE: A Health Research Institute is a powerful strategic commitment to promote biomedical research in hospitals. To assess user satisfaction is an essential quality requirement. The aim of this study is to evaluate the professional satisfaction in a Health Research Institute, a hospital biomedical research centre par excellence. METHODS: Observational study was conducted using a satisfaction questionnaire on Health Research Institute researchers. The explored dimensions were derived from the services offered by the Institute to researchers, and are structured around 4 axes of a five-year Strategic Plan. A descriptive and analytical study was performed depending on adjustment variables. Internal consistency was also calculated. RESULTS: The questionnaire was completed by 108 researchers (15% response). The most valued strategic aspect was the structuring Areas and Research Groups and political communication and dissemination. The overall rating was 7.25 out of 10. Suggestions for improvement refer to the need for help in recruitment, and research infrastructures. High internal consistency was found in the questionnaire (Cronbach alpha of 0.9). CONCLUSIONS: So far research policies in health and biomedical environment have not been sufficiently evaluated by professionals in our field. Systematic evaluations of satisfaction and expectations of key stakeholders is an essential tool for analysis, participation in continuous improvement and advancing excellence in health research.

Disparate antioxidant enzyme activities in cultured human cutaneous fibroblasts, keratinocytes, and melanocytes.

Antioxidant enzyme activities of cultured human foreskin fibroblasts, keratinocytes, and melanocytes from healthy black and Caucasian donors were measured and compared. Fibroblasts had more (p less than 0.05) peroxidase, catalase, glutathione peroxidase, and superoxide dismutase activity than keratinocytes. Keratinocytes had more (p less than 0.05) peroxidase, catalase, glutathione peroxidase, and superoxide dismutase activity than melanocytes. No differences in antioxidant enzyme activities were observed between the cells of any type taken from black or Caucasian people. Antioxidant enzyme activities may affect resistance to damage by oxidants induced by ultraviolet radiation and inflammation.

Successful treatment of incipient graft rejection with donor leukocyte infusions, further proof of a graft versus host lymphohaemopoietic effect.

Graft rejection is a major cause of treatment failure after T-cell-depleted stem cell transplantation (TCD-SCT) and remains a therapeutic challenge. Donor leukocyte infusions (DLIs) have an efficient graft versus host effect, which has been successfully used to treat recipient relapses. We hypothesized that this effect could be exploited to counteract the host versus graft reactions responsible for graft rejection. We report two adult patients with haematological malignancies who underwent sex-mismatched TCD-SCT from HLA-identical sibling donors. Peripheral blood (PB) counts and bone marrow (BM) cellularity were studied on a serial basis. Sequential chimaerism and minimal residual disease analysis were performed by FISH on PB and BM samples as well as on leukocyte lineages (T and B lymphocytes and myeloid cells) purified from PB using immunomagnetic technology. Both patients were diagnosed with incipient graft rejection 2-3 months after engraftment, based on persistently decreasing PB counts and BM cellularity together with the observation of decreasing mixed chimaerism (increasing percentage of recipient cells), mostly in whole PB and T lymphocytes. Both patients were successfully treated with a single DLI (1 x 10(7) CD3+ cells/kg), thereafter achieving normal PB counts and BM cellularity as well as complete chimaerism. Interestingly, the only side effect observed was mild graft versus host disease that did not require treatment. In conclusion, provided that an early diagnosis is made, the graft versus host lymphohaemopoietic effect harboured by immunocompetent donor cells can be successfully used for the treatment of incipient graft rejection.

Quantification of C-ERB-B2 gene amplification in breast cancer cells using fluorescence in situ hybridization and digital image analysis.

Fluorescence in situ hybridization (FISH) allows detection of the intercellular heterogeneity of C-ERB-B2 gene amplification in uncultured breast cancer cells. Nevertheless, because high levels of amplification result in coalescence of signals, direct microscopy quantification is restricted to cells wih low levels of amplification or with dispersed signals. A methodology of digital image analysis, using surface and grey-level FISH signals as parameters that permit a rapid, objective, and accurate estimation of gene copy number, is presented. This procedure is independent of the signal overlapping and results in a more accurate quantification and characterization of tumor cell heterogeneity.

Chimerism quantification after sex-matched BMT: how probable is it to find donor/recipient pairs with distinguishable cells?

Chimerism quantification (CQ) after sex-matched bone marrow transplantation (BMT) is based on the identification of autosomal differences distinguishable at the chromosomal level, such as variations within constitutive heterochromatin between the recipient and the donor. The probability of finding distinguishable recipient/donor pairs at the karyotypic level depends on the frequency of the chromosome variants or morphs in the population, on whether recipient and donor are related, and if so, their kinship relation. We have developed a population genetics-based method that allows the estimation of the percentage of post-BMT CQ expected to be informative using any autosomal polymorphic marker. This method has been developed for the most common transplant situations, such as sibling-matched recipient/donor pairs, haploidentical related (parental/filial) pairs, and unrelated pairs. The method developed was applied to a polymorphism of the pericentromeric region of chromosome 3. This polymorphism becomes evident after in situ digestion with the restriction endonuclease Sau3A, and can be successfully used for CQ. It has been estimated that approximately 59% of the cases of BMT from unrelated donors, 36% of those from sibling donors, and 42% from parental/filial donors, are expected to be distinguishable for post-BMT CQ using this approach.

Restriction endonuclease in situ digestion (REISD) and fluorescence in situ hybridization (FISH) as complementary methods to analyze chimerism and residual disease after bone marrow transplantation.

The efficiency of restriction endonuclease in situ digestion (REISD) with Sau3A to analyze chimerism and residual disease (RD) has been tested before and after an allogenic bone marrow transplant (BMT) in an acute lymphoblastic leukemia (ALL) patient. The combined results obtained with REISD and FISH using the appropriate probes for detecting chromosome rearrangements have proven to be useful for the identification and quantification of both the hemopoietic chimerism achieved after BMT and the RD persistent in the patient. The sensitivity of REISD has been determined to be around 95%, i.e., similar to that obtained by FISH. REISD with Sau3A was particularly useful in the analysis of chimerism since this enzyme revealed the polymorphic status of constitutive heterochromatin in human chromosome 3 and thus allowed discrimination of cells derived from donor and recipient. The method itself seems promising since neither a donor/recipient sex mismatch nor a cytogenetic disease marker are needed for its application.

Lineage-specific chimaerism quantification after T-cell depleted peripheral blood stem cell transplantation.

Patients that receive a T-cell depleted (TCD) hematopoietic stem cell transplantation (SCT) show higher risk of graft failure/rejection and of disease relapse than those that receive unmanipulated grafts. The purpose of the present investigation was to analyze the usefulness of chimaerism quantification in bone marrow (BM), peripheral blood (PB), and leukocyte lineages such as T lymphocytes (CD3+,both CD4+ and CD8+), B lymphocytes (CD19+) and myeloid cells (CD15+), for the early detection of graft failure/rejection episodes and disease relapse after TCD-PBSCT. Two of the ten (2/10) patients included in the study showed stable complete chimaerism (CC). The other 8/10 patients showed decreasing mixed chimaerism (MC) and 7 of them had either graft failure (n = 1)/rejection (n = 3) or disease relapse (n = 3). In two patients relapsed from chronic myeloid leukemia, MC was observed in BM and PB, with higher percentages of autologous cells in BM, as well as in leukocyte lineages, with higher percentages of recipient cells in the myeloid lineage than in lymphocytes. Combined analysis of chimaerism and minimal residual disease allowed early diagnosis of relapse and successful rescue therapy with donor leukocyte infusions (DLI), before the onset of hematological relapse. Chimaerism analysis allowed early diagnosis of incipient graft rejection in 3 patients. These patients showed MC both in BM and PB, with greater percentages of recipient cells in PB. Analysis of leukocyte lineages showed higher percentages of autologous cells in T lymphocytes (mainly CD8+) than in B or myeloid cells. Two of these patients were successfully treated with DLI and recovered normal PB counts and BM cellularity, as well as CC. The graft versus recipient hemopoiesis effect harbored by the donor immunocompetent cells infused seems useful forthe treatment of graft rejection, provided that an early diagnosis is made.

Elevated levels of interferon gamma, tumor necrosis factor alpha, interleukins 2, 4, and 5, but not interleukin 10, are present in recurrent aphthous stomatitis.

OBJECTIVE: To investigate our hypothesis that recurrent aphthous stomatitis (RAS), an inflammatory disease of the oral mucosa, is the result of an abnormal oral mucosal cytokine cascade leading to an enhanced cell-mediated immune response directed toward focal areas of the oral mucosa. DESIGN: Prospective nonrandomized case-control study. SETTING: Academic referral center PATIENTS: For part 1, 21 patients with RAS and 7 control patients; for part 2, 6 patients with RAS and 6 control patients. INTERVENTION: For study part 1, lesional and clinically normal oral mucosal biopsy specimens were obtained during an acute episode (within 72 hours of onset of ulcer) from 21 patients with RAS. Normal oral mucosal biopsy specimens were obtained from 7 healthy individuals, who served as controls. In study part 2, oral mucosal biopsy specimens were obtained from 6 RAS and 6 control patients at 24 and 48 hours after surgical trauma to those sites. MAIN OUTCOME MEASURES: Detection of the following messenger RNA (mRNA) types by use of semiquantitative reverse transcriptase polymerase chain reaction. For part 1, interleukins (IL) 2, 4, 5, and 10, interferon gamma, and tumor necrosis factor alpha were measured. For study part 2, IL-10 and interferon gamma were measured. RESULTS: In part 1, elevated levels of IL-2, interferon gamma, and tumor necrosis factor alpha mRNAs were detected in RAS lesions, consistent with a cell-mediated immune response. The IL-10 mRNA was not increased in RAS lesions. In addition, lower resting levels of IL-10 mRNA were detected in the clinically normal mucosa from patients with RAS, as compared with levels seen in the healthy controls. In part 2, at both 24 and 48 hours following trauma to the oral mucosa, the levels of mucosal IL-10 mRNA remained lower in patients with RAS than those observed in healthy controls, while interferon gamma mRNA levels were higher. CONCLUSION: Failure to suppress the inflammatory reaction initiated by trauma or other external stimuli, likely involving a functional deficiency of IL- 10 in the oral mucosa, appears to be important in the pathogenesis of RAS.

Image processing and analysis of fluorescent labelled cytoskeleton.

Cytoskeleton elements play an important role in cellular processes including normal and pathological conditions or even cell death. Although immunofluorescence microscopy techniques have proved useful to study the cytoskeleton, description and comparative results commonly rely on the direct visual analysis of the targeted cells. We report here an objective and quantitative method to analyze the cytoskeletal pattern of cultured HeLa cells based on image processing and analysis (IPA) of fluorescent labelled cytoskeleton components. Different structural quantifiable characteristics using IPA facilities (densitometric profile, number of branched points, Euler number, convexity, orientation value) allow accurate characterization and differentiation of the three types of fibre nets studied (microtubules, actin microfilament, and cytokeratin intermediate filaments). The application of the analytical methods presented here provides information concerning the organization and modification of cytoskeleton components, and could aid in the further understanding of structural and functional aspects of the cytoskeleton under normal and pathological conditions.