RU2721947C1 - Method for predicting a renal response to an anti-myeloma therapy in the patients with myeloma caste-nephropathy, acute renal damage and the need for dialysis - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, particularly to hematology, namely to the prediction of a renal response to an anti-myeloma therapy in myeloma caste-nephropathy with acute renal damage with the need for dialysis. Prediction is carried out by examining biopsy materials of the affected kidney, wherein staining of at least two biopsy materials of the affected kidney, one of which is stained by Masson, the other immunohistochemically, by examining with the antibody to E-cadherin, followed by quantitative analysis of morphological changes in biopsied affected kidneys by computer morphometry, determining interstitium area, interstitial fibrosis area and E-cadherin expression area, then calculating interstitial fibrosis (FI) index as the ratio of the FI area to the interstitial area ×100, in %, and the E-cadherin expression area index as the E-cadherin expression area to the interstitium area ratio ×100, in %. If the E-cadherin expression area index is less than 10 % in a combination with the interstitial fibrosis area index greater than 40 %, the absence of the renal response to the effective anti-myeloma therapy in the patients with myeloma caste nephropathy, acute renal damage and dialysis need is predicted.EFFECT: invention provides high reliability of the prediction of a renal response to the anti-myeloma therapy in the patients with myeloma caste nephropathy, acute renal damage and the need for dialysis.1 cl, 4 dwg, 1 tbl, 2 ex

Description

The invention relates to medicine, in particular to hematology, and can be used to predict a renal response to anti-myeloma therapy in myeloma castnephropathy with acute renal damage (AKI) with a need for dialysis.

In 13% of patients with multiple myeloma (MM), AKI develops with a need for dialysis due to myeloma caste-nephropathy [Knudsen LM, Nielsen B, Gimsing P, Geisler C. Autologous stem cell transplantation in multiple myeloma: outcome in patients with renal failure. Eur J Haematol 2005; 75: 27-33]. The treatment standard in such situations is three-component bortezomib-containing programs with high doses of dexamethasone, ensuring the achievement of a hematological response in 71% of patients. However, despite the high frequency of the general hematological response, the renal response in dialysis-dependent renal failure does not exceed 30-38% [Rekhtina I.G., Mendeleeva L.P., Varlamova E.Yu., Biryukova L.S. Comparison of the effectiveness of bortezomib-containing programs in achieving an early hematologic and renal response in patients with myeloma nephropathy with dialysis-dependent renal failure. Hematol. and transfusiol., 2015, 4: 4-7]. In order to increase the effectiveness of AKI therapy, attempts have been made to combine chemotherapy with extracorporeal elimination of light chains. However, despite the early initiation of therapy and the rapid reduction of monoclonal light chains, renal function improves only in 33-66% of patients. And in some patients, renal failure is irreversible, despite the most effective methods of treatment. It is important to note that intensive treatment protocols in patients with severe AKI are associated with frequent and severe infectious complications leading to high early mortality. In this regard, the question arises of the justification of such an intensive treatment in all patients, since some of them have irreversible changes in the kidneys.

L, Thierry A, Debiais-Delpech С, Chevret S, Javaugue V, Desport E, et al. Prognostic value of kidney biopsy in myeloma cast nephropathy: a retrospective study of 70 patients. Nephrol Dial Transpl. 2015; 0:1-9]. При этом остается не ясным, что следует относить к распространенному фиброзу интерстиция. По принятой полуколичественной градации, вторая степень включает диапазон от 25% до 50% от площади интерстиция, а 3-я - более 50%. Таким образом, имеющиеся данные противоречивы, их прогностическое значение не изучалось.Despite the fact that morphological changes in the kidneys with myeloma caste-nephropathy are well understood, the criteria for the irreversibility of renal failure are not known. The complexity of solving this problem is due to several reasons. First, the renal response depends on the hematologic response to chemotherapy, which has changed significantly with the advent of targeted drugs. Secondly, a semi-quantitative analysis based on an approximate (visual) assessment remains the standard approach to assessing changes in the kidneys. Some researchers consider widespread interstitial fibrosis and tubular atrophy to be an unfavorable factor, while others consider the number of protein cylinders (30 or more in 10 fields of view) in the lumens of the tubules [

L, Thierry A, Debiais-Delpech C, Chevret S, Javaugue V, Desport E, et al. Prognostic value of kidney biopsy in myeloma cast nephropathy: a retrospective study of 70 patients. Nephrol Dial Transpl. 2015; 0: 1-9]. At the same time, it remains unclear what should be attributed to widespread interstitial fibrosis. According to the accepted semi-quantitative gradation, the second degree includes a range from 25% to 50% of the interstitium area, and the 3rd - more than 50%. Thus, the available data are contradictory, their prognostic value has not been studied.

One of the key pathogenetic mechanisms of the formation of interstitial fibrosis and tubular atrophy is the epithelial-mesenchymal transformation (EMT) of the proximal tubule epithelium. Previous studies have shown the severity and significance of this process in myeloma castnephropathy [Hertig A, Bormard G, Ulinski T, Colombat M, Jouanneau C, Baugey E, et al. Tubular nuclear accumulation of Snail and epithelial phenotypic changes in human myeloma cast nephropathy. Hum. Pathol. 2011.42 (8): 1142-1148]. However, data on the effect of EMT on the possibility of improving renal function have not been studied.

Using digital technology, a computer morphometry technique has become available that allows for accurate quantitative analysis of pathological changes in nephrobiopathy. However, computer morphometry of nephrobioptates of patients with myeloma caste-nephropathy with a separate assessment of all pathological changes has not been previously performed.

Thus, there are currently no prospective multicenter studies of the prognosis of the renal response to anti-myeloma therapy in patients with myeloma caste-nephropathy, acute renal damage, and the need for dialysis. Existing recommendations are mainly based on the experience of individual researchers. Patients with severe AKI pose the greatest problem, since it is in patients of this category that kidney function improvement is less likely to occur, higher early mortality, and shorter overall survival. It is this group of patients that needs to develop new approaches to therapy aimed at quickly achieving the maximum hematological response, which may help improve kidney function in most patients.

Therefore, the identification of predictors of renal response in AKI with the need for dialysis is of great practical importance for the development of individual approaches to therapy, which would avoid the unjustified risk of serious complications.

The technical result consists in the high reliability of the prognosis of the renal response to anti-myeloma therapy in patients with myeloma caste-nephropathy, acute renal damage and the need for dialysis.

The technical result is achieved by predicting a renal response to anti-myeloma therapy in patients with myeloma caste-nephropathy, acute renal damage and the need for dialysis by examining the biopsy samples of the affected kidney, and at least two biopsy samples of the affected kidney are stained, one of which is stained for Masson, another immunohistochemically, examining with an anti-E-cadherin antibody, after which the morphological changes in the biopsy samples of the affected kidneys are quantified by computer morphometry, the area of interstitium, the area of interstitial fibrosis and the expression area of E-cadherin are determined, then the interstitial fibrosis index (FI) is calculated as the ratio of the area of FI to the area of interstitium × 100, in% and the index of the area of expression of E-cadherin, as the ratio of the area of expression of E-cadherin to the area of interstitium × 100,% and when the value of the expression area of E-cadherin is less than 10% in combination with fibrosis area index value and An increase of 40% predicts a lack of a renal response to effective anti-myeloma therapy in patients with myeloma caste-nephropathy, acute renal damage and a need for dialysis.

A prognostic model of the probability of a renal response in myeloma castnephropathy with AKI with the need for dialysis has not been developed.

The method is as follows.

The standard test protocol for biopsy samples of affected kidneys includes the study of at least two biopsy samples of affected kidneys. The obtained biopsy samples are fixed in formalin (10% solution), then an ascending concentration of alcohols is carried out and paraffin embedded. Then prepare sections from paraffin blocks with a thickness of 3-4 microns. Some of them are stained with Masson Trichrome, while others are stained immunohistochemically, examined with antibodies to E-cadherin (clone NCH-38). The immunohistochemical study technique is performed in the classical way. Sections from paraffin blocks, 3-4 microns thick, are mounted on slides coated with poly-L-lysine. After the stage of drying and dewaxing, the antigens are unmasked by the method of high temperature heating. In order to block the appearance of background staining, the glasses are washed in flowing Tris-salt buffer (pH 7) for 10 minutes, washed again with distilled water, preventing the sections from drying out. At the stage of immune staining, primary antibodies are applied to the sections. The antibody titer and incubation time are pre-selected experimentally individually for each antibody. To detect bound antibodies, a Leica polymer-based detection system is used for 30 minutes. Next, the sections are treated with a solution of diaminobenzidine for 1 to 3 minutes and washed in distilled water. Before entering under a coverslip, sections were incubated sequentially in ethanol, then in xylene. At the end, the prepared preparations are enclosed under a coverslip using a special Leica balsamic medium. To determine the expression of proteins, the cortical substance of nephrobioptates is examined at a 100-fold increase.

Quantitative analysis of morphological changes in the biopsy specimens of the affected kidneys is carried out by computer morphometry. The technique consists of several stages and is described in detail in a free-to-access file [http://links.lww.com/TP/A493]. The algorithm used to quantify the area of FI and expression area of E-cadherin is based on the analysis of color digital images obtained by light microscopy using a special optical system and software. We used a Nikon DXM1200 color camera mounted on a Nikon Eclipse E1000 M Microscope (Nikon, Japan), a Zeiss Mirax Scan scanner (Zeiss, Germany) with a 20, NA 0.8 lens and a Marlin CCD camera to export the optical image to digital format (tiff )

The following indicators are determined by the method of quantitative analysis:

1. The area of interstitium, (PI)

2. The area of interstitial fibrosis (PFI)

3. The area of expression of E-cadherin (PE E-cadherin)

4. The index of fibrosis of interstitium,%.

(Ratio of FI area to interstitium area × 100).

5. The index of the area of expression of E-cadherin,%

(The ratio of the area of expression of E-cadherin to the area of interstitium × 100).

For this, the calculation of PI is carried out by digital processing and computer morphometric analysis of the optical image of the cortical substance of the biopsy specimen of the affected kidney stained by Masson, while the area of the cortical substance of the kidney (sKV), as well as its following structures, are sequentially measured: the area of all glomeruli, including sclerosed (sClub), the area of the lumen of the tubules (sPan), the area of the cylinders, including pathological in the lumens of the tubules (sCyl), then the PIs are calculated by the formula: PI = sKB- (sClub + sPan + sPil).

The calculation of PFI is carried out by calculating the area of fibrous pathological tissue in the interstitium throughout the cortical substance of a kidney biopsy, using computer morphometry to automatically select the affected area stained with Masson aniline blue trichrome (blue) according to the specified color range, after which automatic calculation is carried out the area of the entire selected area.

Calculation of the expression area of E-cadherin is carried out by calculating the entire expression region of the kidney biopsy specimen of the antibody expression zone, using computer morphometry - automatically highlighting this zone, immunohistochemically colored in brown, according to the specified color range, after which the area of the entire selected area is automatically calculated .

Based on the obtained data, the interstitial fibrosis index is calculated,%, as the ratio of PFI to PI × 100 and the expression area index of E-cadherin,%, as the ratio of PE E-cadherin to PI × 100.

The examination included 25 patients with myeloma castnephropathy and AKI with a need for dialysis, in whom a hematological response was achieved as a result of induction chemotherapy. The diagnosis of myeloma castnephropathy was established on the basis of light and immunofluorescence microscopy of kidney biopsy specimens. In all patients, a biopsy of the affected kidney was performed before the start of anti-myeloma therapy. In addition to the main indicators of the interstitial fibrosis index,% and the expression area index of E-cadherin,%, a study was made of indicators of the number of sclerotic glomeruli,%, (the ratio of the number of sclerotic glomeruli to the total number of glomeruli × 100); the number of protein cylinders in 1 field of view (the ratio of the number of protein cylinders in all fields of view to the number of fields of view); the area index of cellular inflammatory infiltration,% (the ratio of the area of cell infiltration to the area of interstitium × 100); vimentin expression area index,% (ratio of vimentin expression area to interstitium area × 100); index of expression area of alpha-smooth muscle actin,% (ratio of expression area of alpha-smooth muscle actin to interstitium area × 100). The median time from AKI diagnosis to performing a kidney biopsy was 14 days (0-73). Of the 25 patients, a renal response was achieved in 17 (independence from hemodialysis), and 8 did not have a renal response. Using computer morphometry, a quantitative analysis of pathological changes in the kidney biopsy was performed. The results of the study are presented in table 1.

Of all the analyzed parameters, statistically significant differences were revealed by the indices of the expression area of E-cadherin and the area of FI.

To determine the significance of the FI indicator determined by computer morphometry, a ROC analysis was performed as a prognostic marker for the probability of a renal response (see Fig. 1). The resulting curve allows us to conclude that the quantitative indicator of FI has good reliability of the prediction of the renal response, taking into account the value of the area under the curve. Thus, this diagnostic feature can be used in a prognostic assessment.

In FIG. Figure 1 shows the ROC curve for predicting renal response by interstitial fibrosis index. PHI - interstitial fibrosis. Values above the ROC curve are the estimates of the likelihood of a renal response corresponding to the selected FI threshold. The area indicator under the curve (0.79) indicates the reliability of the prognosis of the renal response according to the FI values.

The method of ROC analysis also proved the high reliability (p <0.05) of the prevalence of E-cadherin expression in predicting the renal response (see Fig. 2). In FIG. Figure 2 shows the ROC curve for predicting the renal response by the area expression index of E-cadherin in nephrobiopathy. The area under the curve is 0.75 (significantly higher than 0.5), which indicates good reliability of the prognosis of the renal response by the numerical values of the expression of E-cadherin. The numerical values above the ROC curve are the estimates of the probability of a renal response corresponding to the selected threshold for the expression of E-cadherin.

Correlation analysis did not reveal a statistically significant relationship between the expression area indices of E-cadherin and PI (Rs = -0.335, p = 0.065).

When these two most significant factors were combined, the area under the ROC curve increased to 0.84 (each symptom individually had 0.79 and 0.75), which indicates an increase in the accuracy of the prognosis of the renal response or its absence (see Fig. 3 ) In FIG. Figure 3 shows the ROC curve for predicting the renal response by two morphometric parameters (the index of the area of fibrosis of interstitium and the index of the area of expression of E-cadherin). The area under the curve is 0.84 (significantly higher than 0.5), which indicates a very good reliability of the prognosis of the renal response by the numerical values of the area of interstitial fibrosis and the expression of E-cadherin. Numerical values above the ROC-curve - assessment of the probability of a renal response corresponding to the selected measurement threshold.

Further, using the method of two-dimensional discriminant analysis, a prognostic model was developed for two signs: the area of the PI and expression of E-cadherin. Their threshold values are determined - for the E-cadherin area index less than 10%, for the FI area index more than 40%. Based on what a logical rule has been created (a new complex feature) that defines an unfavorable group for the renal response among all patients: the renal response is not achieved with a probability of 93.3% if the E-cadherin area index is less than 10% and the FI area index is more than 40% (odds ratio (OR) = 24.5).

In FIG. Figure 4 shows a model for predicting a renal response in patients with myeloma caste-nephropathy with AKI with the need for dialysis. Characteristic of this rule. If the patient is assigned to an unfavorable group, then his likelihood of a lack of a renal response to effective anti-myeloma therapy is 24 times greater than that of a patient assigned to a favorable group. Of the patients who were in an unfavorable group by the expression area indices of E-cadherin and FI, only 6.67% (1 out of 14 people) received a renal response as a result of anti-myeloma therapy.

The expression area of the epithelial marker E-cadherin is less than 10% in combination with an area of interstitial fibrosis of more than 40% of the interstitium area - prognostic factors for the absence of a renal response in the effectiveness of anti-myeloma therapy in patients with myeloma castnephropathy with acute renal damage with a need for dialysis.

Examples of the method

Example 1

Patient P., 70 years old. Multiple myeloma in this patient debuted with the development of acute renal damage (blood creatinine 960 μmol / L, oliguria 300 ml / day, GFR <5 ml / min), and therefore, the patient was started on emergency hemodialysis. For diagnostic purposes, a kidney biopsy was performed, the results of which revealed myeloma castnephropathy. Clinical diagnosis: Multiple myeloma, proceeding with B-J-kappa paraproteinemia, stage III by R-ISS. Myeloma caste-nephropathy. Acute renal damage stage L (RIFLE classification); Concomitant disease: Coronary heart disease: postinfarction cardiosclerosis. Paroxysmal form of atrial fibrillation. Circulatory failure PA stage. In a semi-quantitative morphological study, the severity of FI was defined as II degree. Biopsy specimens of affected kidneys were examined by light microscopy stained with Masson's aniline blue trichrome and immunohistochemical staining and studies with antibodies to E-cadherin described above. Quantitative analysis of morphological changes in biopsy specimens of affected kidneys by computer morphometry showed the following: interstitium area (PI), microns = 3832391.6; the area of interstitial fibrosis (PFI), microns = 1366241.8. Hence, the interstitial fibrosis index,% is 1366241.8 / 3832391.6 × 100 = 35.6%.

Area of interstitium, (PI), μm = 3832391.6; the area of expression of E-cadherin (PE E-cadherin), μm = 552235.2. The index of the area of expression of E-cadherin was 552235.2 / 3832391.6 × 100 = 14.4%.

Based on the model presented by us, the patient belongs to the favorable renal response to anti-myeloma therapy. We have carried out treatment according to the standard 3-component XT regimen (bortezomib, cyclophosphamide, dexamethasone), without dose reduction. Treatment was complicated by the development of bilateral pneumonia, a recurrent rhythm disturbance. As a result of therapy after 2 courses received a hematological response. After 1.5 months from the start of treatment, renal function improved, and renal replacement therapy was completely abandoned.

Thus, the prediction of the renal response influenced the choice of therapy. The restoration of renal function and the cessation of hemodialysis justifies the choice of intensive care tactics, despite the development of severe complications in an elderly patient with severe concomitant pathology.

Example 2

Patient M., 58 years old. Against the background of the development of severe acute renal damage (blood creatinine 1500 μmol / L, GFR <5 ml / min) hemodialysis was urgently started. For diagnostic purposes, a biopsy of the affected kidneys was performed, and a diagnosis of myeloma caste-nephropathy was established. Subsequently, the diagnosis of multiple myeloma was confirmed. In a morphological semi-quantitative study, the degree of expression of FI corresponded to 2 degrees, i.e. indefinite interval for predicting renal damage reversibility. Biopsy specimens of affected kidneys were examined by light microscopy stained with Masson's aniline blue trichrome and immunohistochemical staining and studies with antibodies to E-cadherin described above. Quantitative analysis of morphological changes in kidney biopsies by computer morphometry showed the following: interstitium area (PI), microns = 2417793.8; area of interstitial fibrosis (PFI), μm = 1308802.5. Hence, the interstitial fibrosis index,% is 1308802.5 / 2417793.8 × 100 = 54.1%.

Area of interstitium, (PI), μm = 2417793.8; the area of expression of E-cadherin (PE E-cadherin), μm = 213795.4. In this case, the expression area index of E-cadherin was 213795.4 / 2417793.8 × 100 = 8.8%.

Based on the prognostic model, the absence of a renal response to effective anti-myeloma therapy is predicted; renal damage is irreversible even with a quick and effective chemotherapeutic effect.

The patient underwent 2 courses of chemotherapy according to the standard bortezomib-containing program (bortezomib, cyclophosphamide, dexamethasone), as a result of which a deep hematological response (OChR) was achieved. Treatment was complicated by the development of bilateral pneumonia, sepsis, which required a transfer to the intensive care unit. Despite the rapid achievement of a deep hematological response, renal function has not improved (and in the future too), on the basis of which the terminal stage of chronic kidney disease with the need for hemodialysis has been ascertained.

The use of a prognostic model made it possible to establish an extremely low probability of a renal response. More appropriate tactics in this case would be to conduct chemotherapy in a less intensive mode, which would avoid the development of life-threatening complications.

Claims (1)

A method for predicting the renal response to anti-myeloma therapy in patients with myeloma caste-nephropathy, acute renal damage and the need for dialysis is carried out by examining biopsy samples of the affected kidney, and at least two biopsy samples of the affected kidney are stained, one of which is stained according to Masson, the other is immunohistochemically, examining with an antibody to E-cadherin, after which a quantitative analysis of morphological changes in the biopsy samples of the affected kidneys is carried out by computer morphometry, determine the area of interstitium, the area of fibrosis of interstitium and the expression area of E-cadherin, then calculate the index of fibrosis of interstitium (FI) as the ratio of FI to the interstitial area × 100, in%, and the E-cadherin expression area index as the ratio of the E-cadherin expression area to the interstitium area × 100,%, and when the E-cadherin expression area index is less than 10% in combination with the value of the interstitial fibrosis area index over 40% predict from the absence of a renal response to effective anti-myeloma therapy in patients with myeloma caste-nephropathy, acute renal damage and the need for dialysis.

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