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ORIGINAL ARTICLE  
Year : 2021  |  Volume : 64  |  Issue : 2  |  Page : 323-328
Primary cutaneous amyloidosis: A clinicopathological, histochemical, and immunohistochemical study


Department of Pathology, JSS Medical College, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India

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Date of Submission21-Jan-2020
Date of Decision27-Apr-2020
Date of Acceptance08-Jun-2020
Date of Web Publication9-Apr-2021
 

   Abstract 


Background: Primary cutaneous amyloidosis (PCA) comprises several forms of localized cutaneous amyloidosis characterized by amyloid deposits occurring at or near dermal–epidermal junctions. Immunohistochemical studies have shown the expression of cytokeratin (CK) suggesting that it has an epidermal origin. Objectives: To study the clinicopathological features of PCA and expression of CK5/6 and correlate it with Congo red stain. Materials and Methods: A total of 30 histologically proven cases of PCA were studied. Congo red staining and immunohistochemical expression of CK5/6 were analyzed. Statistical Analysis: The qualitative data has been expressed as proportions and the quantitative data has been expressed as mean ± SD. All data was analyzed using the Statistical Package for Social Sciences (SPSS) software version 22. Results: Deposits of amyloid in papillary dermis were seen in all 30 cases. Mild focal basal cell vacuolar degeneration and apoptotic bodies in epidermis were seen in six cases. The presence of pigment cells in dermis were seen in 26 cases. CK5/6 showed weak/mild immunopositivity in nine cases, moderate in 20 cases, and strong in one case. Conclusion: The presence of dermal melanophages interspersed within eosinophilic deposits gives a clue to the diagnosis. Congo red stain highlights the deposits and visualization under polarized light gives apple green birefringence which is diagnostic of amyloid. Staining of amyloid deposits by CK5/6 proves that the amyloid is of keratinocyte origin. There was 100% sensitivity with Congo red and CK5/6. Thus, CK5/6 can be used as an adjunct tool to Congo red stain in the diagnosis of primary cutaneous amyloidosis.

Keywords: Amyloidosis, Congo red, immunohistochemistry

How to cite this article:
Sinha A, Manjunath G V, Basavaraj V. Primary cutaneous amyloidosis: A clinicopathological, histochemical, and immunohistochemical study. Indian J Pathol Microbiol 2021;64:323-8

How to cite this URL:
Sinha A, Manjunath G V, Basavaraj V. Primary cutaneous amyloidosis: A clinicopathological, histochemical, and immunohistochemical study. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 May 16];64:323-8. Available from: https://www.ijpmonline.org/text.asp?2021/64/2/323/313280





   Introduction Top


Amyloids are extracellular proteinaceous deposits which are resilient to proteolytic digestion and has distinguishing physical characteristics. In primary localized cutaneous amyloidosis (PLCA), the accumulation of amyloid arises in normal appearing skin, with no evidence of deposits in internal organs.[1] Amyloid has characteristic physiochemical properties like Congophilia and apple green birefringence when visualized under polarized light.

Electron microscopic studies have shown that some of amyloid material in primary local forms of cutaneous amyloidosis derived from the epidermal cells through filamentous degeneration.[2] Immunohistochemical studies have shown expression of cytokeratin in the amyloid deposits suggesting that it has an epidermal origin.[3]

Also, the result of immunohistochemical studies have shown that CK5/6 is the major cytokeratin present in the amyloid deposits of PCA.[4]


   Materials and Methods Top


A prospective and retrospective observational study was conducted at the Department of Pathology and Dermatology, JSS Medical College and Hospital, Mysuru over a period of four and half years from January 2015 to July 2019. The skin biopsies of all clinically suspected cases of primary cutaneous amyloidosis which were proven histopathologically were included in the study. Inadequate biopsies and congo red negative samples of suspected primary cutaneous amyloidosis cases were excluded from the study.

A total of 30 histopathologically diagnosed cases of primary cutaneous amyloidosis were studied. The clinical findings were retrieved from the records for retrospective study. Along with relevant clinical history of the patients, 3 to 5 mm punch biopsy of the representative skin lesions of the patients were received. The histopathology slides were analyzed and repeat histologic sections were taken from the paraffin blocks and were stained with hematoxylin and eosin (H&E), congo red stain (Qualigen) and studied under polarized light (Olympus Bx 41) for apple green birefringence. The primary antibodies of CK5/6 were applied to the sections.

Immunohistochemical staining

Immunohistochemistry (IHC) was performed on 3- to 4-μm-thick sections on poly-l-lysine coated slides. The slides were baked at 60°C for 1 hour in hot air oven. Slides were deparaffinized, rehydrated, and heated in a pressure cooker containing antigen retrieval solution and sodium citrate buffer at pH 6.

One liter of the retrieval solution was brought to boil in the pressure cooker. The slides were placed in metal staining racks and lowered into pressure cooker ensuring that the slides were completely immersed in the retrieval solution. When the pressure cooker reached the operating temperature and pressure, it was timed for 15 minutes or up to 2 to 3 whistles. The pressure cooker was removed from the heat source and cooled by placing it under running cold water with the lid on. The slides were cooled and washed with water and buffer solution. A peroxide block was applied for 10 min and washed with Tris buffered saline (TBS) for 2–5 min. The protein block was applied for 10 min and washed with TBS for 2–5 min. The sections were incubated with the primary antibody Antibody: CK5/6(DAKO) for 1 hour and washed with TBS for 2–5 min. Then, post primary block/enhancer was applied for 30 min and washed with TBS for 2–-5 min. The sections were incubated with SS label (polymer) for 30 minutes and washed with TBS for 2–5 min. The bound antibody was visualized using a DAB-chromogen substrate which was prepared by adding 50 μl of DAB Chromogen to 1 ml of DAB buffer. The sections were rinsed in running water and counterstained with hematoxylin and again rinsed in water for 5 min. The external positive control tissue included the sample of normal skin biopsies.

In negative controls, primary antibody was omitted and phosphate buffer saline was substituted.

Method of reporting by IHC

The primary antibodies of CK5/6 were applied to the sections. The grading was given based on the number of specks of brown-colored granular staining in the sections.

None - 0

Weak/Mild - + (1–5 specks)

Moderate - ++ (6–10 specks)

Strong/- +++ (10–20).

Statistical analysis

All data were analyzed using the Statistical Package for Social Sciences (SPSS) software version 22. The mean, standard deviation, frequency, and percent were employed in the present study. The Chi -square test procedure tabulates a variable into categories and computes a Chi-square statistic.


   Results Top


Thirty-eight skin biopsies with the clinical diagnosis of amyloidosis were received in the Department of Pathology between January 2015 and July 2019. Thirty cases showed amyloid deposits on histopathology and were included in the study. The skin biopsies were analyzed histopathologically and by immunohistochemical staining for CK5/6.

Twenty-two cases (73.3%) were histopathologically diagnosed as lichen amyloidosis and eight cases (26.7%) were diagnosed as macular amyloidosis. These were confirmed by congo-red staining under polarized light. No cases of nodular amyloidosis were noted in the study.

The majority of cases were seen in the 4th–5th decade with a female preponderance of 66.7% and male-to-female ratio of 0.6:1. The age range was between 18 years to 86 years with a mean age of 45 ± 17.5 years. The duration of lesions ranged from 12 months to 37 months. Pruritis was the presenting symptom in 14 cases (46.7%) followed by the hyperpigmented patch in 12 cases (40%). Sixty six percent of the patients gave the history of friction with towels, sponges, and pumice stone. The most common site affected was lower limbs followed by back and forearm. Patients presented with papules in 22 cases (73.3%) and with macules in eight cases (26.7%).

The histopathological examination of H and E stained sections revealed epidermal orthokeratosis, acanthosis, papillomatosis, and elongation of rete ridges. There were small globular deposits of amorphous eosinophilic acellular material in the papillary dermis along with mild perivascular lymphocytic infiltration [Figure 1]. Those cases with epidermal changes were characterized as lichen amyloidosis and those with unremarkable epidermis were characterized as macular amyloidosis.
Figure 1: (a) Photomicrograph showing acanthotic epidermis with deposits of amyloid in the papillary dermis (H&E, x 100). (b) Photomicrograph showing apoptotic bodies (blue arrow) in epidermis and pigment cells amidst amyloid (yellow arrow) in dermis (H&E, x 400)

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All the 30 cases showed orange-red deposits on congo red stain [Figure 2]. The existence of amyloid was confirmed by polarized light which showed apple-green birefringence [Figure 3].
Figure 2: Photomicrograph showing orange-red deposits of amyloid in papillary dermis (Congo red stain, x 200)

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Figure 3: Photomicrograph showing apple-green birefringence of the amyloid deposits under polarized light (Congo red stain under polarized light, x 400)

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Apart from amyloid deposits, the study was undertaken to see additional epidermal and dermal findings associated with cutaneous amyloidosis like the presence of basal cell vacuolar degeneration and apoptotic bodies in epidermis, presence of pigment cells and plasma cells in dermis along with the presence of thin bands of compressed collagen separating amyloid deposits from epidermis [Figure 1].

The deposits of amyloids in papillary dermis were seen in all 30 cases. Mild focal basal cell vacuolar degeneration and presence of apoptotic bodies in epidermis were seen in six cases. The presence of dermal melanophages in dermis were seen in 26 cases.

All the 30 cases showed positivity with CK5/6. The intensity of immunoreactivity was graded as weak/mild, moderate, and strong. Grading was given based on the number of specks of brown color granular staining in the sections.

None - 0

Weak/Mild - + (1–5 specks)

Moderate - ++ (6–10 specks)

Strong - +++(10–20 specks).

Weak/mild positivity was seen in nine cases, moderate in 20 cases, and strong in one case [Figure 4].
Figure 4: (a) Photomicrograph showing CK5/6 Mild +immunopositivity having 1–5 specks (x 400). (b) Photomicrograph showing CK5/6 Moderate ++immunopositivity having 6–10 specks (x 400). (c) Photomicrograph showing CK5/6 Strong +++immunopositivity having 10–20 specks (x 400)

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   Discussion Top


The first description of amyloid was given by Rokitansky and Budd in 1842.[5] Amyloidosis is a condition related with a numeral of inherited and inflammatory disorders in which extracellular deposits of fibrillar proteins are accountable for tissue damage and functional compromise. Amyloid deposits are considered as homogenous, amorphous, eosinophilic, and acellular material upon hematoxylin and eosin staining.

Special stains are required to demonstrate amyloid. Congo red is the most confirmatory test which gives an orange-red staining reaction to these deposits. The amyloid deposits showed apple-green birefringence when seen under polarized light. The staining characteristics are the result of the cross β pleated sheet conformation of the polypeptide backbones of the amyloid fibril.[6]

Amyloidosis is classified as systemic and localized.[6] PCA typically comprises macular amyloidosis, lichen amyloidosis, and nodular or tumefactive amyloidosis. The macular and papular forms may coexist in the same patient and this is known as biphasic amyloidosis.[1] In the present study, there were no cases of nodular amyloidosis.

In the progression of amyloid deposition, there is filamentous disintegration and apoptosis of basal keratinocytes trailed by the conversion of filamentous masses (or colloid bodies) into amyloid material in the papillary dermis.[7]

According to the studies done by Vijaya B et al., Y. T Chang et al., S. Sumitra et al., and Aysun Uguz et al., the male preponderance was seen in the presentation of primary cutaneous amyloidosis while in the present study female preponderance was seen with the male to female ratio of 0.6:1.[1],[4],[8],[9] Cutaneous amyloidosis is a chronic process and the duration of the lesions ranged from 12 months to 37 months. Pruritis is the most common symptom which was present in half of the cases in present study. Clinically 66.6% of the patients had a history of friction with towels, sponges, and pumice stone which play a role in precipitating primary cutaneous amyloidosis. Friction has been observed to be one of the associated findings in cutaneous amyloidosis.[8] The lesions most commonly occur in the lower limbs, back, and forearm in the descending order of frequency as observed in the present study. Patients presented with papules in 22 cases (73.3%) and with macules in eight cases (26.7%). Similarly, studies by Vijaya B et al., Wang et al. also demonstrated lichen amyloidosis to be the most common form of PCA.[1],[10]

In 1928, Gutmann described lichen amyloidosis.[1] It is the most common type of primary cutaneous amyloidosis and is found mostly in Chinese ancestry people, South east Asia and South America.[9] Its etiology is unknown but chronic pruritis has been proposed as an etiological factor. A working theory states that due to long-term scratching and rubbing, there is epidermal trauma which results in the degradation of keratinocytes and development of amyloid. It typically appears as red-brown, hyperkeratotic, pruritic papules.[6] Histopathologically, the lesions reveal orthokeratosis, acanthosis with elongation of rete ridges, and mild papillomatosis. In the present study, we also looked for apoptotic bodies and focal basal cell vacuolar degeneration and it was seen in six cases accounting to 20%. Basal cell vacuolar degeneration and the presence of apoptotic bodies were observed in the present study which has also been documented in the literature by various authors in the study on cutaneous amyloidosis.[1],[8],[9],[11] The presence of apoptotic bodies and basal cell vacuolar degeneration indicates that the derivation of cutaneous amyloid deposits could be from degenerated keratin peptides of apoptotic keratinocytes due to dermal macrophages and fibroblasts. The presence of pigment cells observed amidst the amyloid deposits may be attributed to basal cell vacuolar degeneration.[12] In the present and the other studies, the majority of the cases showed pigment cells.[1],[8],[9],[11] These dermal melanophages could be secondary to pigment incontinence due to basal cell vacuolar degeneration.

The amyloid deposits were seen as amorphous, globoid deposits of acellular eosinophilic material in the expanded papillary dermis. The dermis also showed mild perivascular lymphocytic infiltration.

Macular amyloidosis is seen in India, Middle East, and Central and South America.[9] It presents as small, gray-brown macules which may blend together to produce hyperpigmented patches. These hyperpigmented macules or patches are found on the upper back and less frequently on the chest or extremities which may have a characteristic rippled or reticulated pattern and which tend to continue unchanged for many years.[13] Histopathological changes of macular amyloidosis were similar to lichen amyloidosis. The quantity of amyloid deposits was less compared to lichen amyloidosis and there were no marked epidermal changes.

All the 30 cases demonstrated acellular eosinophilic homogenous deposits in the dermis which upon staining with congo red showed the deposits of orange-red substances. It was visualized under polarized light microscopy which showed apple-green birefringence which confirmed the presence of amyloid.

In comparison with other studies, the sensitivity for congo red staining in the detection of amyloid was 100% which was similar to the present study [Table 1].
Table 1: Comparison of positive congo red-stained slides with other studies

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The amyloid may be seen with several stains, including methyl violet, crystal violet, thioflavin T, and congo red. The most common staining techniques used is congo red staining, as amyloid shows a characteristic apple-green birefringence when viewed under polarized light microscopy. Although H and E staining may indicate for amyloid diagnosis, but congo red staining under polarized light has proved to be sensitive and definitive.[13]

Various techniques are available to demonstrate amyloid. The direct evaluation of the fibril type is done by immunohistochemistry and proteomics. Fibril sequencing is accessible today to recognize the type of amyloid, although the gold standard is the congo red staining.[14]

Cytokeratin are intermediate filament proteins which are expressed in the cytoplasm of epithelial cells.[7] Amyloid mainly comprises of cytokeratin CK5; therefore, anti-CK5/6 are antibodies useful for its recognition.[13] Cytokeratin 5, 1, 10, and 14 are the major components of amyloid in lichenoid and macular amyloidosis.

Immunohistochemical studies have shown the expression of cytokeratin CK1-8, CK5/6/18, CK14, CK10, and CK34βE12 in the amyloid deposits. It suggests that amyloid has an epidermal origin.[15],[16],[17],[18],[19],[20] In the diagnosis of PCA, immunostaining for cytokeratin has evidenced to be extremely superior to congo red. In the present study, 30 cases stained for CK5/6 of cutaneous amyloidosis were analyzed and all the 30 cases showed positive immunoreactivity to antibodies CK5/6. The patterns of intensity of immunoreactivity by CK5/6 have been correlated with other studies [Table 2].
Table 2: Comparison of immunohistochemistry CK5/6 with other studies

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In the present study, CK5/6 reacted with amyloid deposits in all 30 cases which is similar to the study done by R Apaydin et al.[7] He observed immunoreactivity to CK5/6 antibodies in 13 cases (eight cases of lichen amyloidosis and six cases of macular amyloidosis). There was no difference in the staining characteristics of cytokeratin between lichen and macular amyloidosis. R Apaydin et al. also observed immunopositivity in 10 cases with CK1-8 and there was no immunoreactivity with CK10, CK14, CK17, CK18, CK8/18, and CK19.[7] A study done by Inoue et al. showed immunopositivity for antibody 34βE12 in all 20 cases.[16] Another study by Huilgol et al. showed immunopositivity with CK5 in lichen and macular amyloidosis which was similar to the present study.[21] Therefore, it suggested that CK5/6 might be involved as the common precursor in formation of amyloid.

Immunohistochemical staining of amyloid deposits revealed that monoclonal anticytokeratin antibodies are often reactive, suggesting their epidermal origin. In the present study, 100% positivity for CK5/6 was seen which showed that immunostaining can be used to confirm the existence of amyloid deposits.


   Conclusion Top


The diagnosis of cutaneous amyloidosis involves clinical, histological, and immunohistochemical analyses. Lichen amyloidosis is the most common variant of primary cutaneous amyloidosis followed by macular amyloidosis. Pruritis and friction play a very important part in the progression of cutaneous amyloidosis.

Cutaneous amyloid deposits are seen as amorphous acellular eosinophilic material in papillary dermis. Although H and E staining helps to identify amyloid as an amorphous eosinophilic substance, congo red stain highlights the deposits and visualization under polarized light gives a apple-green birefringence which is diagnostic of amyloid.

The presence of dermal melanophages interspersed within eosinophilic deposits gives a clue to the diagnosis. The staining of amyloid deposits by CK5/6 proves that the amyloid is of keratinocyte origin. There is 100% sensitivity with congo red and with CK5/6.

CK5/6 can be used as an alternative tool to Congo red stain in the diagnosis of primary cutaneous amyloidosis. The present study supports the utility of CK5/6 for diagnosis of cutaneous amyloidosis.

Recommendations

  • Not many studies have been done correlating the clinicopathological features, histochemical staining, and immunohistochemical features. So, this study is an attempt to correlate and understand the pathogenesis of cutaneous amyloidosis and to establish immunohistochemical marker as a tool to understand the pathogenesis of amyloid in primary cutaneous amyloidosis
  • Amyloid substance showed positive staining with cytokeratin CK5/6 in all the cases and therefore it may be used as a substitute tool to Congo red staining wherever the facility of polarized microscopy is not available.


Limitations

  • The primary limitation of the current study was the small sample size although it involved the data for four and half years
  • Immunohistochemical studies have shown the expression of various cytokeratins such as CK1-8, CK5/6/18, CK14, CK10, and CK34βE12 in the amyloid deposits whereas in the present study only CK5/6 was used for the confirmation of amyloid deposits.


Acknowledgement

The authors sincerely thank the Department of Dermatology for their constant support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Vijaya Basavaraj
Department of Pathology, JSS Hospital, Mysuru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_32_20

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