[Multiple myeloma: response to treatment and survival of patients according to the interim analysis of the Russian observational, retrospective-prospective, multicenter cohort study (MULTISPECT)].

AIM: The primary objective of the interim analysis of the MULTISPECT study was to evaluate the short-term efficacy of the treatment and long-term outcomes in cohorts of primary and pretreated patients with multiple myeloma (MM) receiving treatment in actual clinical practice in various regions of the Russian Federation. Secondary objectives were a description of the main characteristics of patients; analysis of the most commonly used therapy regimens of the 1st and later lines and the sequence of their changes; evaluation of the response to therapy. Additional objectives included evaluation of the effect of the new COVID-19 coronavirus infection on the course of MM in patients. MATERIALS AND METHODS: The study is an observational retrospective-prospective multicenter cohort study. For its implementation, a structured database of patients with MM was used, provided by hematologists of the centers affiliated for the study. RESULTS: The study included 1,294 patients (cohort 1 806, cohort 2 488). In both cohorts, patients aged 6069 years were in the majority. 3 lines of therapy (L1, L2, L3) were used for cohort 1; in cohort 2, the 4th line of therapy was also used in 2 patients. The therapy regimens were analyzed for 290 (22.41%) of all patients in the study. Responses to therapy were analyzed for 214 patients of cohort 1 and 109 patients of cohort 2. Autologous and allogeneic hematopoietic stem cell transplantations were carried out for a limited proportion of patients in both cohorts. At the end of the study and upon presentation of its results, the status of patients was the following: 96% of patients in cohort 1 and 89% in cohort 2 were alive. The therapy regimens in both cohorts were characterized by variability. The most commonly used regimens in each of the lines of therapy have been identified. The most used therapy regimen in patients with MM of both cohorts was the VCD-regime. Rd-regime in cohort 1 and RD-regime in cohort 2 were the second most frequent used regimens. In patients of both cohorts, the therapy regimens including Bortezomib were most often used. CONCLUSION: The variety of therapy regimens used to treat MM in actual clinical practice may be due to the factors of availability of new medicines and updated recommendations for the treatment of the disease. Further, in the context of this study, a more detailed analysis of the efficacy of certain therapy regimens in the 1st and later lines on progression free survival and overall survival of MM patients should be carried out.

Comparative Analysis of the Pharmacological Activity of Bis(3,5-di-Tert-Butyl-4-Hydroxyphenylthiolate)Dimethylol in Different Modes of Administration on a Mouse Model of Melanoma B16 Tumor Growth.

We performed a comparative analysis of the pharmacological activity of the hybrid organotin compound bis(3,5-di-tert-butyl-4-hydroxyphenylthiolate)dimethylol (Me-3) administered in different modes to mice with transplanted melanoma B16 to identify the most effective dosage regimen. Three modes of administration were used: preventive (before transplantation of tumor cells), classical according to Z. P. Sof'ina (48 h after transplantation of tumor cells), and delayed (7 days after transplantation of tumor cells, after formation of nodules of the primary tumor node). Compound Me-3 was administered at a total dose of 375 mg/kg intraperitoneally once a day for 5 days. The classical mode of administration was identified as the most effective, which indicates the preventive antimetastatic activity of Me-3 on the model of the transplanted mouse tumor melanoma B16.

Effect of Hybrid Organotin Compound on Activity of LPO and Antioxidant Protection of the Liver Tissues in Animals with Melanoma B16.

The central element of the "metastatic organotropism" is a shift of the pro/antioxidant balance in cells and activation of oxidative stress and protective antioxidant systems. We studied the effects of bis(3,5-di-tert-butyl-4-hydroxyphenylthiolate)dimethylol (Me-3) in the maximum effective and toxic total doses on the level of markers of oxidative stress and antioxidant protection in the liver of mice with melanoma B16 before the appearance of macroscopic metastases. In 48 h after tumor inoculation, Me-3 was administered intraperitoneally once a day for 5 days in total doses of 375 and 500 mg/kg according to the classical method. Administration of the hybrid organotin compound Me-3 produced different effects on the pro/antioxidant state of the microenvironment of liver tissue as the target of melanoma B16 metastasis. The results suggest that inversion of the anti/prooxidant profile of Me-3 is determined by its dose.

[Monoclonal gammopathy of renal significance: consensus of hematologists and nephrologists of Russia on the establishment of nosology, diagnostic approach and rationale for clone specific treatment].

Monoclonal gammopathy of renal significance (MGRS) is a new nosology in modern nephrology and oncohematology. MGRS is defined as kidney injury due to nephrotoxic monoclonal immunoglobulin produced by the B-cell line clone which does not reach the hematological criteria for specific treatment initiation. Monoclonal proteins pathological effects on kidney parenchyma result in irreversible decline of kidney function till the end stage renal disease that in line with the position of International Consensus of hematologists and nephrologists determinates critical necessity for clone specific treatment in patients with MGRS despite the absence of hematological indications for treatment initiation. Main challenge of MGRS in Russian Federation is an inaccessibility of an in-time diagnostic and appropriate treatment for the great majority of patients due to the following reasons: 1) limited knowledge about the MGRS among hematologists and nephrologists; 2) lack of necessary diagnostic resources in most health-care facilities; 3) lack of approved clinical recommendations and medical economic standards for treatment of this pathological entity. Consensus document comprises the opinion of experts leading nephrologists and hematologists of Russian Federation on the problem of MGRS including the incoherence in nosology classification, diagnostics approach and rationale for clone specific treatment. Consensus document is based on conclusions and agreements reached during the conference of leading nephrologists and hematologists of Russia which was held in the framework of symposia Plasma cell dyscrasias and lymphoproliferative diseases: modern approaches to therapy, 1516 of March 2019, Pavlov First Saint Petersburg State Medical University. The present Consensus is intended to define the principal practical steps to resolve the problem of MGRS in Russian Federation that are summarized as final clauses.

[The treatment of patients with circulatory system pathology in health resorts "Tarkhovskii" and "Priozerskii"].

The authors showed the history of foundation and development of health resorts "Tarkhovsky" and "Priozersky", as well as an analysis of the treatment of patients with circulatory system diseases in these health resorts. It was found that the structure of patients fitted the profile of health resorts and in recent years has remained stable. Patients with circulatory system diseases accounted for 47.8%. The existing system of medical rehabilitation and restorative treatment of patients with diseases of the circulatory system in the health resorts "Tarkhovsky" and "Priozersky" ensures the implementation in full rehabilitation and recovery programs for all categories of contingent attached.

[The use of a "buffet" style meals in the system of health-resort treatment].

The authors summarized literature sources, concerning modern ideas about the role of nutritional therapy in the system of health-resort treatment. The authors also made a research on the basis of literature, concerning health-resort nutrition divided into two systems-- buffet system and special-order system. On the basis of sanatorium establishments of the Ministry of Defense authors summarized an experience of clinical nutrition in different age groups and categories of patients. On the basis of current regulatory acts were defined forms and methods of meal catering in the Russian Federation. The results of the study are a scientific supposition for substantiation of management decision-making process, which allows to increase an efficiency of health-resort treatment via nutrition therapy.

Hyperimmunoglobulinemia E and efficacy of elimination diet in two patients with Schnitzler syndrome.

BACKGROUND: Schnitzler syndrome (SS) is a rare clinical entity characterized by chronic recurrent urticarial rash, monoclonal IgM gammopathy, intermittent fever and other symptoms. In this report, we present the cases of two patients with SS: a male and a female aged 50 and 49 years, respectively. Both patients had hyperimmunoglobulinemia E and showed good response to elimination diet. METHODS: The patients had chronic urticaria, IgM gammopathy and an elevation of the serum levels of inflammation markers. Total IgE levels were found to be high (2000 U/ml and 540 U/ml, respectively). No underlying causes for hyperimmunoglobulinemia E (allergy, parasites, etc.) were revealed. The first patient did not respond to the treatment with antihistamines, while the second one responded only to high doses. The response to prednisolone in the second patient was incomplete. RESULTS: Following a strict elimination diet resulted in marked improvement in skin lesions in both patients. In one of our patients we observed a decrease in IgE and IgM levels after a 3 week diet. The systemic symptoms persisted and improved only after adding pefloxacin, followed by a 3-day empirical course of intravenous prednisone in the first patient and a course of plasmapheresis in the second one. CONCLUSION: The high serum levels of total IgE may be associated with chronic urticaria activity, severe disease course and a poor response to treatment with antihistamines, and may be considered a possible marker of a subset of patients with SS showing a good response to the restriction diet. In general, we can assume that elimination diet can have an influence on the skin lesions and other symptoms of SS as well as on total IgE and IgM levels, but such association, the underlying mechanisms and the reasons for excessive IgE synthesis should be investigated in further studies.

[The prognostic value of evaluation of minimal residual disease using technique of flow cytofluorometry during application of therapy of chronic lymphatic leukemia].

The achievement of molecular remission is associated with increasing of survival of patients with chronic lymphatic leukemia. The important direction of research is seeking of parameters applicable to forecast of response to therapy. The purpose of the study was evaluating prognostic significance of indicator of minimal residual disease detected by technique of flow cytofluorometry of peripheral blood of patients with chronic lymphatic leukemia during therapy application. The sampling included 112 patients with chronic lymphatic leukemia aged from 43 to 82 years. All patients were given treatment consisted of 6 courses of immune chemotherapy combined with fludarabine with cyclophosphan and rituximab. The samples of peripheral blood were analyzed after 3 courses during therapy and after 6 courses after completion of treatment. The cells were analyzed using 5 and 6 color flow cytometry for the purpose of detection of immune phenotype associated with chronic lymphatic leukemia. The evaluation of minimal residual disease was implemented according international standardized protocol (Rawstron A.C. et al. 2007; 21 (5): 956-64). The minimal residual disease negative status was reached in 87 (78%) patients during evaluation of response after 6th course of treatment. The implementation of indicators of residual disease after 3 courses with fludarabine, cyclophosphan and rituximab permitted to sort out two groups of patients with chronic lymphatic leukemia i.e 67 patients with low (< 0.12%) level of minimal residual disease and 45 patients with high (> 0.12%) level of tumor cells. The rate of molecular remission after completion of treatment. in the given groups consisted 100% and 44% correspondingly. The study demonstrates possibilities of early immune phenotype evaluation of minimal residual disease to forecast differences in response to treatment in patients with chronic lymphatic leukemia that makes it possible to avoid undesirable toxicity of therapy or to choose method of consolidation.

[The characteristics of evaluation of expression of ZAP-70 in tumor cells under b-cell chronic lymphatic leukemia using the flow cytofluorometry technique].

The b-cell chronic lymphatic leukemia is the most common among all lymphatic proliferative diseases and is characterized by significant variability of its clinical course. The mutation status of genes of variable region of heavy chains of immunoglobulins (IgVH) is the most reliable prognostic factor forecasting time until beginning of treatment in case of b-cell chronic lymphatic leukemia. However its detection nowadays is inaccessible for routine diagnostics. Among surrogate markers of mutation status the indicator of expression of ZAP-70 by tumor cells estimated using flow cytofluorometry. However, in publications there are different guidelines concerning the technique of mentioned marker. To establish the optimal approach to evaluation of expression of ZAP-70 the peripheral blood samples of 5I patients with b-cell chronic lymphatic leukemia and 10 healthy persons were analyzed. The comparison with the results of detection of mutation status of IgVH-genes revealed the advantage of applying the technique of calculation of MFI ratio during interpretation of data of expression of ZAP-70 obtained with flow cytofluorometry. In this framework, the indicator of expression of ZAP-70 can be applied in assessing the course of disease and time until the beginning of treatment of b-cell chronic lymphatic leukemia.

[The detection of minimal residual disease in patients with chronic B-cell lymphatic leukemia using patient-specified polymerase chain reaction].

The new effective protocols of treatment of chronic B-cell lymphatic leukemia, including purine analogs and monoclonal antibodies, provide robust remissions under this disease. Accordingly, the requirements to remission quality assessment are changed too. In particular the assessment of minimal residual disease is obligatory. To assess minimal residual disease in terms of quantity in case of chronic B-cell lymphatic leukemia the technique of polymerase chain reaction was applied in real time with patient-specific primers from the area of V-D-J combinations of genes of heavy chain of immunoglobulin. The study included samples from 60 patients suffering of chronic B-cell lymphatic leukemia. In 15 of them (25%), it was impossible to apply neither the sequence analysis of genes of heavy chain of immunoglobulin nor the fitting of patient-specific primer. The results of quantitative determination of minimal residual disease were obtained in 45 patients (55 tests). The minimal residual disease was detected in 30 of 55 samples (54.5%) and was not detected in 25 of 55 samples (45.5%). At the same time, the quantitative determination of minimal residual disease was implemented in regard to the initial level of neoplastic cells. The method sensitivity qualified by serial dilutions, consisted 10(-5) or 1 neoplastic cell to 100 000 normal cells. The comparative analysis was applied to the results of determination of minimal residual disease using two methods -polymerase chain reaction in real time using patient-specified primers and four-color flow cytofluometry. The determination of minimal residual disease with both methods was implemented in 37 patients (45 tests). The results of both methods matched in 93.3% (42 tests out of 45) with maximal disparity of one degree. Then Spearman factor consisted 0.87 (p < 0.0001). In 3 out of 45 tests (6.7%) neoplastic cells were detected with only one method. In the first case, it was the method of four-color flow cytofluometry and in other two cases it was polymerase chain reaction in real time. Therefore, the detection of minimal residual disease under chronic B-cell lymphatic leukemia using the method of polymerase chain reaction in real time is rather sensitive and specific and correlates with the results received with the method of four-color flow cytofluometry. The results are the same in the case of using anti-CD20 monoclonal antibodies under treatment.

[PCR definition of B-cell clonality: which electrophoretic method is better].

AIM: To compare diagnostic efficacy of B-cell clonality determination in application of different electrophoretic methods. MATERIAL AND METHODS: B-cell clonality was determined with different techniques in 89 patients having B-cell lymphoma (n = 48), B-cell lymphoma in remission (n = 11), reactive processes (n = 24), lymphogranulomatosis and T-cell lymphoma (n = 6). Healthy donors served control. Clonality was defined by rearrangements of Ig heavy chain genes with usage of primers to FR2 region of Ig gene. Electrophoretic methods were the following: agarose electrophoresis and SSCP. RESULTS: Clonality was detected in the presence of more than 5.9% clonal cells from total count of mononuclear cells or more than 20% of clonal cells from total number of B-lymphocytes in the sample. We achieved detectability of B-clonality in B-cell tumors 87.5% (42 of 48 cases). False negative tests were also investigated with kits of primers to FR1 and FR3 regions, but clonality was not determined. Among reactive processes, non-B-cell tumors and healthy donors clonality was found in 1 of 41 cases. This patient had acute respiratory viral infection. Agarose electrophoresis and SSCP test results coincided. CONCLUSION: Determination of B-cell clonality with agarose electrophoresis proved to be a simple, reliable, cost-effective and reproducible method of differential diagnosis of tumor and reactive lymphoproliferation. This method is not suitable for verification of tumor remission or assessment of minimal residual disease because of its low sensitivity.

[Paraneoplastic lymphadenopathy].

AIM: To study a spectrum of histologic patterns in patients with paraneoplastic lymphadenopathies, to analyse repeating changes and the causes of diagnostic mistakes. MATERIAL AND METHODS: In a retrospective analysis of 457 patients observed in Hematology Research Center of the RF in 1994-2004, and diagnosed as having non-malignant lymphadenopathies, we identified 40 patients in whom the second or third biopsy showed lymphoma. Nineteen patients (47.5%) had Hodgkin's lymphomas, 11 (27.5%)--B-cell lymphomas and 10 (25%) T-cell lymphomas. RESULTS: In patients subsequently diagnosed with Hodgkin's lymphoma there were 4 repeating histologic patterns in paraneoplastic lymph nodes: sinus histiocytosis (7 patients, 37%), paracortical reaction with numerous plasma cells and macrophages (7 patients, 37%), marked fibrotic changes (4 patients, 21%) and necrotizing lesions (3 patients, 16%). In 15 patients the second biopsy was performed within 4 weeks, while in 4 patients intervals between biopsies were 2, 4, 10 and 24 months. Eight patients (42%) had paraneoplastic changes in lymph nodes located not in the adjacent zones (in 7 patients above the diaphragm, and in 1 patient in both sides of the diaphragm). Amongst patients with B-cell lymphomas 7 had follicular lymphomas, 3--diffuse large B-cell lymphomas and 1--mantle cell lymphoma. In paraneoplastic lymph nodes there were 3 cases of sinus histiocytosis, 1--necrosis. In 5 patients with follicular lymphoma initially diagnosed as having follicular hyperplasia, retrospective analysis and immunohistochemistry showed partial involvement of lymph nodes with lymphoma. In two of them the presence of malignancy was clinically evident at the moment of the first biopsy, while three had a long history of lymphadenopathy (time to diagnostic biopsies were 5, 13 and 34 months). Amongst patients with T-cell lymphomas 5 had undetermined peripheral T-cell lymphomas, 2--anaplastic large cell lymphomas, 1--angioimmunoblastic lymphoma, 1--hepatolienal lymphoma and 1--Lennert's lymphoma. Median of time to the second biopsy was 4 months, range 1-48 months. Five patients (50%) had in the first lymph node sinus histiocytosis, 3 (30%)--paracortical hyperplasia, and 2--granulomatous lesions. Clonal rearrangements of gamma-chain T-cell receptor genes were found in 2 patients from 3 tested. CONCLUSION: Histologic patterns in lymph nodes not involved by lymphomas in patients with lymphomas are not random. While sinus histiocytosis and necrosis are universal findings, some patterns are disease specific. Paracortical hyperplasia is typical for T-cell lymphomas, prominent fibrosis--for Hodgkin's lymphoma. We believe that paraneoplastic changes in lymph nodes are caused by partial tumor involvement, cytokine reaction, or antitumor immune response. From practical point of view, finding of necrosis, prominent sinus histiocytosis, or prominent fibrosis of a lymph node in the absence of a history of chronic lymphadenitis or inflammation in the draining area should be considered as possible indication to second biopsy. Interpretation of such paraneoplastic phenomena as paracortical hyperplasia and formation of epithelioid-cell granulemas is not easy and must consider context of a clinical picture.

[Prognostic significance of immunoglobulin variable region gene mutations in B-CLL patients treated with combination therapy fludarabine plus cyclophosphamide].

AIM: To study prognostic factors in previously untreated patients receiving FC regimen (fludarabine plus cyclophosphamide). MATERIAL AND METHODS: We conducted a retrospective analysis of B-CLL patients observed in Hematology Research Center of Russia (Moscow) and Faculty Therapy Clinic of St. Petersburg State Medical University (St. Petersburg). All patients received FC regimen as a first line treatment (fludarabine 50 mg plus cyclophosphamide 250 mg/m2 for 3 days intravenously, repeated every 28 days). RESULTS: 54 patients were included into the study. The median age was 57.5 yrs (range 40-78 yrs). There were 38 males and 16 females. Before the treatment 22% patients had Binet stage A, 41%--stage B and 37%--stage C. 62% patients had unmutated subtype of B-CLL and 38% mutated subtype. 12 patients (22%) received less than 4 cycles of chemotherapy. In 8 patients (15%) there were significant delays between cycles (more than 2 months). In the whole cohort the median overall survival calculated from the time of treatment initiation was 57.4 months, the median progression free survival--24 months, and the median relapse free survival--27 moths. Mutational status of immunoglobulin variable region genes significantly influenced survival. In patients with unmutated subtype the median progression free survival was 23.6 months, while in patients with mutated subset it was not reached: 75% survival at 22.7 months (p = 0.027). Difference in progression free survival by stages (A versus B+C, A+B versus C) was not significant. CONCLUSION: Our data show that mutational status of immunoglobulin variable region genes remains a significant prognostic factor in patients receiving combined therapy with cyclophosphamide and fludarabine.

Expression level of lipoprotein lipase and dystrophin genes predict survival in B-cell chronic lymphocytic leukemia.

Mutational status of immunoglobulin variable region genes (VH-genes) is known as the strongest predictor of long term prognosis in B-CLL. However, applications in the routine clinical practice are time consuming, and therefore some other predictions are required. In this study, we have compared prognostic values of real time PCR quantification of the expression levels of four genes previously shown to be differentially expressed in V(H)-unmutated and mutated B-CLL subtypes: ZAP-70, ZBTB20, DMD and LPL. The study included 134 B-CLL patients. Expression levels of LPL and DMD genes were significantly correlated to mutational status, while expression levels of of ZAP-70 gene correlated only in CD19+ selected cases (N = 40). No correlation was observed for ZBTB20 gene. Expression levels of LPL and DMD predicted overall survival in the entire cohort of patients. Prognostic values of LPL gene expression levels were significant even for CLL patients with stage A. Quantitative RT-PCR assays for measuring LPL gene expression are robust enough to be introduced into routine clinical practice.

[Determination of T-cell clonicity by a T-cell receptor gamma-chain: final data].

AIM: To test diagnostic efficacy of T-cell clonicity determination by a gamma-chain of T-cell receptor (TCR). MATERIAL AND METHODS: The examination covered 426 patients (458 tests). T-cell tumors were detected in 132 patients. The samples from 294 patients in whom T-cell tumors were not found were referred to the laboratory for a differential diagnosis. Clonicity was determined by gamma-chain of TCR in the test for conformation polymorphism of one-chain DNA fragments. All the tests were made in one laboratory. RESULTS: Sensitivity of the method, found by analysis of different delusions of the cell line Jurkat in selected polyclonal CD3+ cells is 10%. The results were of 3 kinds: clonal, doubtful and polyclonal. In patients free of T-cell tumors there were 15 (5%), 34 (11%) and 258 (84%) false positive, doubtful and true negative results. False positive results were most frequent in an acute phase of infectious mononucleosis--in 8 (33%) of 24 patients. 127 (84%) true positive, 5 (3%) doubtful and 19 (13% 0 false negative results were documented in patients with T-cell lymphoma. The occurrence of false negative results was the highest in anaplastic CD30+ T-cell lymphomas--in 6 (46%) of 13 cases. CONCLUSION: Diagnostic efficacy of the method is 92%, but in 10% the result is doubtful. Main reason of false negative results is a small number of tumor cells in tissue samples. The main reason of false positive results is prevalence of one or some T-cell clones in the presence of immune response caused by viruses, autoimmune diseases and, possibly, depletion of bone marrow in aplastic syndromes.

[Cytogenetic disorders in chronic B-cell lymphoid leukemia and their relations with clinicobiological features and prognosis of the disease].

AIM: To study a relationship between cytogenetic disorders, clinicobiological characteristics and prognosis in chronic B-cell lymphoid leukemia (B-CLL). MATERIAL AND METHODS: Cytogenetic examination of blood, bone marrow and lymph node cells from 135 patients (90 males and 45 females aged 23-84 years) with chronic B-CLL was made. The patients were followed up from 1 month to 25 years. Before the cytogenetic examination specific therapy was not given. B-CLL was staged by K. Rai, forms--by A.L. Vorobyev and M.D. Brilliant. All the patients have undergone standard cytogenetic examination, FISH with multicolor probe to loci with possible frequent aberrations (del3q14, del11q23, del17p13, trisomia 12), determination of CD38 antigen expression on circulating tumor cells. Mutation status of the genes of immunoglobulins variable region (IgVH) was defined in 61 patients. RESULTS: Del13q14 was detected in 34 cases, del11q23--in 26, trisomia of chromosome 12--in 17 cases, del 17p13--in 8, absence of q-arm of chromosome 13--in 3 cases. 61 patients had no karyotype defects. Three prognostic groups of the patients were identified: favourable prognosis--patients without disorders of karyotype and one chromosomal aberration--del13q14; intermediate prognosis patients with dell1q23 and trisomia of chromosome 12; poor prognosis--patients with del17p13 and complex disorders of karyotype. CONCLUSION. Cytogenetic study help determine prognosis of B-CLL and detect patients in need of early therapy.

[The role of morphological characteristics of lymph nodes in differential diagnosis of reactive lymphadenopathies].

AIM: To analyse causes of reactive lymphadenitis regarding histological changes in the lymph node, of diagnostic errors; to determine frequency of establishment of nosological diagnosis. MATERIAL AND METHODS: A retrospective analysis of diagnostic examination of 375 patients with reactive lymphadenitis observed from 1992 to 2000 was made. RESULTS: For differential diagnosis 6 groups of reactive lymphadenopathies with different histological picture were identified: follicular hyperplasia, paracortical hyperplasia, granulomatous lymphadenitis, granulomatous pyonecrotic lymphadenitis, sinus histiocytosis as a leading sign and node necrosis as a leading sign. The analysis of making nosological diagnosis for each group and of diagnostic errors was conducted. CONCLUSION: Characteristics of lymph node changes in reactive lymphadenitis is of great importance. Better cooperation between histologists and clinicians must improve efficacy of reactive lymphadenitis diagnosis.