Mycosis fungoides (MF) is a primary epidermotropic T-cell skin lymphoma (SL) with a typical proliferation of small and medium-sized T-cells, some of which have cerebriform nuclei. A classic variant of the MF course is the staged evolution of spots, plaques, and nodules. MF slowly progresses over several years or even decades. In later stages, lymph nodes and internal organs may be involved. The diagnosis of MF is based on a comprehensive assessment of the clinical presentation and histological and immunophenotypic (IPT) examination of biopsy specimens from skin lesions [1–5].

At the morphological examination, the patchy (erythematous) stage is characterized by epidermotropic superficial small focal perivascular infiltrates, the plaque stage by epidermotropic dense band-like infiltrate in the upper part of the dermis, the tumor stage by dense focal or diffuse infiltrates, involving the entire dermis and penetrating subcutaneous fatty tissue; epidermotropism may be absent. MF is characterized by an infiltrate of α/β-T-helper cells, mature memory cells with the following immunophenotype: βF1⁺ CD3⁺ CD4⁺ CD5⁺ CD7⁺ CD8^– CD45RO⁺ [5–9].

The ISLE-EORTC guidelines distinguish the early stages of MF: IA, IB, and IIA. The diagnosis of these stages is based on cutaneous manifestations (4–5 points), with no or moderate blood involvement (0–1 points) and lymph node involvement only in stage IIA (1–2 points). Clinical presentation at early stages includes localized spots, papules, and/or plaques covering less than 10% of the skin, with spots only at stage IA, spots and forming single plaques at stage IB, and spots and single plaques at stage IIA [9–13].

In most cases, MF is detected at stage II, with the appearance of plaques in patients' skin [12, 13]. At the Urals Research Institute of Dermatovenerology and Immunopathology, 69 cases of GM were identified during 2010–2018 based on clinical, pathomorphological, and morphometric studies performed. These cases accounted for 14.1% of examined patients with suspected skin lymphoproliferative diseases. Stage I (erythematous) MF was detected in 14.5% of patients, stage II (plaque) in 76.8%, and stage III (tumorous) in 8.7% [14].

Skin biopsy is a valuable technique widely used for diagnosis and follow-up in dermatological practice. In dermato-oncology, a skin biopsy is essential since it is the only method for subsequent tumor verification. Skin biopsy is indicated in unclear clinical diagnosis of dermatosis and skin neoplasms with suspected malignancy [13, 15].

The most challenging issue in oncodermatology is detecting the early (erythematous) stage I MF, characterized by the spots on the skin. Skin changes and pathomorphological abnormalities in stage IA MF are minimal, and clinical presentation at the onset of this disease is the same as in chronic dermatoses (CD: atopic dermatitis, eczema, parapsoriasis, psoriasis) [4, 16]. The examination of histology specimens stained with hematoxylin-eosin commonly shows moderate paravasal infiltrates in the papillary layer of the dermis with local epidermotropism with preserved epidermis at this stage of the disease. Monoclonal antibody (MAb) kits (panels) have been developed and recommended for diagnosing primary skin lymphoma, determining the disease type, and immunophenotype of lymphocytic/lymphoid proliferative elements. The selection of the antibody and reagent panels for differential diagnosis of various diseases is driven by the objectives of the test [5, 17, 18].

The morphometric study can objectify pathomorphological data and monitor the changes in the lymphoid infiltrate parameters, including during therapy. The number of histological sections examined should comply with the guidelines for morphometric studies and reflect a true picture of the disease [19, 20].

Currently, there are no unified diagnostic criteria for MF in foreign publications, and clinical guidelines vary considerably regarding diagnostic tests [5, 10, 13]. There is no published evidence for a differentiated pathomorphological pattern typical for the early MF stages (IA, IB, and IIA). Single morphometric studies can reliably identify early and late stages of MF [14, 21].

The objective of the study is to identify infiltrate formation features in the dermis of patients with MF stages IA, IB, and IIA and determine quantitative criteria of lymphoproliferative process severity.

Materials and methods

The study was based on data from the clinic of Urals Research Institute of Dermatovenerology and Immunopathology obtained in 2018–2021. Clinical and pathomorphological studies were carried out to establish the diagnosis of MF at early stages in 30 patients: erythematous stage in 18, erythematous-plaque stage in 12.

The incisional method of skin biopsy with partial or complete excision of the affected skin area with 5–7 mm in length, 2–3 mm in width, and 3–4 mm in height of the excised block was used. This method allows the sampling of large enough biopsy specimens with minimal mechanical damage, containing the necessary biological material for further pathomorphological, immunohistochemical (IHC), and quantitative studies.

Pathomorphological studies were performed on the light-optical level using paraffin sections of 4–5 μm thick stained with hematoxylin-eosin and IHC slides made in automatic mode using IHC staining system LEICA BOND-MAX (Germany). The selection of the antibody and reagent panels for diagnosis of lymphoproliferative diseases was driven by the objectives of the test and included 9 MAbs: CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30, and the proliferating cell marker Ki-67.

A DM 4000 B laboratory microscope (Leica) was used to examine histologic specimens and IHC slides. The images were entered using a hardware-software complex including research-level light microscope Axio Imager M2, digital camera and specialized software ZEN 2012 (ZEISS, Germany), a computer unit with sufficient RAM, and a monitor with high resolution and color rendering.

Morphometric studies were performed on specimens from 27 patients using IHC slides. Immunophenotyping was performed using CD3 MAbs to determine the severity of T-cell proliferation; these cells are the main substrate of the dermal infiltrates in MF. For quantitative research, SIAMS Photolab software (Russia) was used to determine the volume fraction (VF) of the test item (CD3+ cells positive staining area) in each of the examined visual fields with the calculation of the mean volume fraction (MVF) and the standard deviation (SD). For each case, five standard visual fields of 5,002,624 μm2 with the most pronounced lymphoproliferation were examined. The total examined area for each specimen was 25,013,120 μm2. A total of 150 images were studied. The obtained data are presented in reports in tables, histograms, and microphotographs of the examined areas.

Results and discussion

The pathomorphological studies allowed to observe different manifestations of lymphoproliferation in the skin of MF patients, clinically defined as early stages (IA, IB, and IIA) [5, 13].

The intensity of lymphoid infiltration in the epidermis and dermis was minimal in 9 of 30 examined patients (group 1). In these observations, the pathomorphological pattern corresponded to the description in the previously mentioned guideline [5], i.e., epidermotropic superficial perivascular infiltrates were observed. The preservation of the epidermis with a small number of lymphocytes in the basal layer was revealed. The dermal papillae were minimally filled with CD3⁺ cells, with only single lymphocytes, as well as in the dermis between the epidermis and the infiltrate. Typically, the aggregates of CD3⁺ cells were observed in the walls and near microvessels in the upper dermis. In addition, infiltration of the vessel wall by CD3⁺ cells could be observed in the deeper layer of the dermis (Fig. 1).

Fig. 1. Typical histoarchitecture of infiltrate and CD3⁺ MVF values in 5 investigated skin areas of group 1 patient with minimal manifestations of lymphoproliferative process.

Patient K., 41 years old. Immunophenotyping method, ×400.

In the excised skin fragment of group 1 patients, the visually and quantitatively detectable intensity of the lymphoproliferation was insignificant. The VF of cells in the infiltrate was not more than 0.100. Thus, in the dermis of patient K. VF values of CD3⁺ cells ranged from 0.033–0.064 (see. Fig. 1). The MVF for this group of patients was 0.058, and the SD was 0.014 (see Table).

CD3⁺ MVF and SD values in the groups of patients (n=27) with MF at early stages

A fundamentally different pathomorphological pattern was observed in the skin biopsy of group 2 patients (n=12): CD3⁺ lymphocytes were more densely located in the vascular walls and more intensively filled the paravasal spaces of the subpapillary dermis. The formation of a relatively dense and extensive infiltrate was observed in some areas. In the areas with the most pronounced lymphoproliferation, the adjoining infiltrate to the epidermis was observed, in which a significant number of CD3⁺ cells were present in basal and spinous layers (Fig. 2).

Fig. 2. Typical histoarchitecture of infiltrate and CD3⁺ MVF values in 5 investigated skin areas of group 2 patient.

Patient A., 57 years old. Immunophenotyping method, ×400.

Significant differences in the infiltrate histoarchitecture of group 2 patients were also confirmed by the results of morphometric studies. Thus, in patient A., the VF of CD3⁺ cells was similar to that in group 1 only in three sites; in two sites, it was 0.095 and 0.169, significantly exceeding the values in group 1. The VF variability in the patient was significant; the range was 0.029–0.169. The MVF in group 2 patients was 0.136, and the SD was 0.024 (see Table).

Group 3 (n=9) showed the most pronounced changes in both the epidermis and dermis of the patients. The formation of extensive and relatively dense infiltrates in both subpapillary and papillary layers of the dermis was observed in most of the examined areas. The areas with the highest intensity of lymphoproliferation were the most closely adherent to the epidermis. Most of the VF values of CD3⁺ cells were not in the similar range as in groups 1 and 2; in particular, in patient Shch., it was in the range of 0.208–0.263 (Fig. 3).

Fig. 3. Typical histoarchitecture of infiltrate and CD3⁺ MVF values in 5 investigated skin areas of group 3 patient.

Patient Shch., 81 years old. Immunophenotyping method, ×400.

The morphometric studies performed on the images presented in Fig. 1–3 also provided different statistical criteria (SMV, SD, and distribution histograms) of lymphoproliferation severity in these patients. Thus, in patient K., whose morphological pattern included the previously described minimal changes in the papillary and subpapillary layers of the dermis, minimal values, and variability of MF (see Fig. 1), the columns in the distribution histogram were separated insignificantly; the MVF was 0.047, and the SD was 0.013 (Fig. 4, A).

Fig. 4. Typical distribution histograms in early MF.

a — group 1 (patient K., 41 years old), b — group 2 (patient A., 57 years old), c — group 3 (patient Shch., 81 years old).

The least pronounced pathomorphological changes and minimal values of studied parameters of lymphoproliferation severity shown by morphometric studies indicate the beginning of the disease and correspond to the stage IA of MF.

In the more severe lymphoproliferation observed in patient A., the histogram shows minimal values in only 3 of the 5 images studied, but the MA on the other images (2 of 5) is outside the range typical for group 1 and significantly exceeds these values. The variability of VF values in the examined areas reflects the obtained MVF and SD values. In addition, the SD has maximum values exceeding half of the MVF (see Fig. 4, B). We believe the results of this patient specimen examination may indicate the transition of MF from stage IA to stage IB.

Figure 3 shows infiltrate histoarchitecture in five examined investigated skin areas of patient Shch. differs significantly from previous cases in the most intense lymphoproliferation, similar to described in the clinical guidelines stage II (plaque) MF: epidermotropic dense band-like infiltrate in the upper dermis [5, 13]. The histogram in Fig. 4, C contains only one site out of five examined with a range of 0.090–0.170, typical for the previous observation; the remaining sites have a range of 0.170–0.260. The MVF was 0.213, and a relatively high SD value of 0.054 was obtained. This observation may demonstrate stage IIA MF.

The obtained most typical quantitative criteria, MVF and SD for each of the three groups consisting of 9 observations of early stages of GM, are presented in the Table.

The small SD values in all three groups, ranging from one-tenth to one-fourth of the MVF values, confirm that the observations are in the early stages of MF.

The results of clinical, pathomorphological, and morphometric studies demonstrate the MF progression in its early stages. Visually observed changes in the skin from a single spot to single plaques, as well as transformations of the histoarchitecture of the infiltrate in the dermis from minimal lymphoproliferation in stage IA MF to the formation of an extensive and denser infiltrate in stage IIA MF (see Fig. 1–3) correlate with the quantitative results, namely a gradual increase in CD3⁺ cells within the infiltrate in the early stages of MF.

Thus, clear differences in the pathomorphological pattern in patients with early stages of MF, observed visually, are confirmed by the results of morphometric studies. The obtained data supplement and objectify the knowledge on the course of lymphoproliferation in the derma, clinically defined as the erythematous stage. The possibility of detecting the stages IA, IB, and IIA of MF by visual assessment of skin and histological specimens during comprehensive examination will contribute to the improvement and optimization of diagnostics of early stages of this primary skin lymphomas (GM), which is an urgent and one of the most challenging issues in oncodermatology.

Conclusion

A comprehensive study shows differences in the intensity of lymphoproliferation at the beginning of the disease (stage IA) and after some time (stages IB and IIA). When using IPT-method and morphometric studies, it is possible to elaborate more precise criteria of the pathomorphological pattern of differentiated diagnosis of early stages of MF. Diagnosing the early stages of malignant diseases is one of the most urgent issues of modern medicine since patients' survival and quality of life depend on the timely detection of malignancies and proper treatment.

Authors’ contributions:

The concept and design of the study: N.V. Kungurov, G.D. Safonova, M.M. Kokhan

Collecting and interpreting the data: G.D. Safonova, O.G. Rimar, I.A. Kuklin

Statistical analysis: G.D. Safonova

Drafting the manuscript: G.D. Safonova

Revising the manuscript: M.M. Kokhan, I.A. Kuklin

The authors declare no conflict of interest.