Tag: Ki-67

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jhc.2024.v8.i2.5


Editorial
Original Contribution
Correlation of Ki-67 Proliferating Index with Histological Types and Characterization of Mucin in Colorectal Carcinoma
*Sultana S,1 Islam N,2 Kabir E3

  1. *Dr. Sahela Sultana, MBBS. MD, Assistant Professor, Department of Pathology, Ibrahim Medical College, Dhaka. sultana.sahela83@gmail.com
  2. Dr. Nasimul Islam. MBBS. M. Phil., Professor & Head, Department of Pathology, Anwar Khan Modern Medical College & Hospital
  3. Dr. Enamul Kabir. MBBS. M.Phil. MSc(Path), Professor, Department of Pathology, Popular Medical College, Dhaka

*For Correspondence
Abstract
Background: Colorectal carcinoma is a major cause of cancer associated with a high rate of morbidity and mortality in the western world. One of the pathologic features considered to be important for prognosis is mucin production. Many authors confirmed that colon carcinomas with high mucin content tend to recur locally and carry a poor prognosis.
Aim: Correlation of Ki-67 proliferating index with different type of colorectal carcinoma as well as characterization of mucin.
Method: This cross sectional study was conducted at Sir Salimullah Medical College, Department of pathology   from July 2014 to June 2016. Ninety eight patients with colorectal carcinoma was enrolled in this  study  who underwent surgical resection of colon, adenocarcinomas. For histological classification  we used the WHO recommendation (2000) and to be more accurate we sub-classified mucinous adenocarcinomas by morphometrical  analysis in three categories: pure mucinous, with extracellular mucin more than 80% of the tumoral volume; mixed type, with 50–80%  extracellular mucin; and mixed type with less than 50% extracellular mucin and their  correlation with Ki-67 proliferating index . For histochemical investigation, we used stains such as: D- PAS and Alcian Blue. A technique of manual tissue array was employed to see Ki-67 expression by IHC method. Ki-67 is a proliferation associated nuclear antigen which can be recognized by MIB-1 monoclonal antibody.
Result: It was observed that Ki-67 labeling index was high in nonmucinous tumor  compared to mucinous adenocarcinoma and signet ring cell carcinoma which is  statistically significant (P<0.05). Histochemical stain of mucin where both D-PAS and Alcian Blue positive cases(mixed type)  are more than the Only D-PAS positive cases(pure type). Ki-67 proliferating index was also high in mixed type mucinous adenocarcinoma (<50%) compared to pure (>80%) and mixed type (50-80%). The result was statistically significant (p<0.05). Correlation of Ki-67 proliferating index with histologic type as well as mucin characterization and thereby provide information to clinician to better understanding  of the  treatment as well as prognosis.

[Journal of Histopathology and Cytopathology, 2024 Jul; 8 (2):100-108]
DOI: https://www.doi.org/10.69950/jhc.2024.v8.i2.5
Keywords: Colorectal carcinoma, Mucin, Immunohistochemistry, Ki-67.
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jhc-2019-v-3-n-2-Is-histopathological-kabir-an

Review Article

Is Histopathological  Examination Essential  for the Diagnosis of Psoriasiform Dermatitis?

*Kabir AN,1 Rahman MM 2

  1. *Dr. AKM Nurul Kabir, Associate Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh. kabir56@gmail.com
  2. Mohammad Mosiur Rahman,  Assistant Professor, Department of Pathology, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh

 *For correspondence

Abstract

Psoriasiform dermatitis encompasses a wide range of diseases, some of which show both clinical and histological overlap. Psoriasis is generally thought to be a genetic disease that is triggered by environmental factors. Psoriasis is characterized by an abnormally excessive and rapid growth of the epidermal layer of the skin.  Abnormal production and an over abundance of skin cells result from the sequence of pathological events in psoriasis, can be shown as a higher Ki-67 index compared with normal appearing, non-lesional skin. Morphometric analysis  of  histological features can also give a quantitative dimension  to histopathology  in diagnosis of psoriasis and in differentiating from other psoriasiform dermatitis. Psoriasis is also regarded as a T-cell mediated disorder, mainly CD4+ (helper/inducer) lymphocytes along with CD8+ subsets are known to occur.  The patient often prove to be a diagnostic dilemma for both dermatologists and pathologists alike. However, clinical features when considered alone may not be reliable, as they vary with both disease duration and treatment.  Though to give a precise diagnosis one relies on clinical correlation, histopathology is essential as it is possible to assign specific diagnosis in most cases with a logical and systematic histopathological approach. Morphometry along with the help of Ki-67 & Cyclin D1 and other immunostains of keratinocytes, and immunophenotyping of T-cell infiltrate, a definitive diagnosis can be made.

 [Journal of Histopathology and Cytopathology, 2019 Jul; 3 (2):151-161]

Key words: Psoriasis, Psoriasiform dermatitis, Morphometry, Ki-67, CD4+, CD8+

 Introduction

Psoriasiform  dermatitis  encompasses a wide range of inflammatory dermatoses, some of which show both clinical and histological overlap.  The term psoriasiform means  that  the lesion either clinically or histopathologically mimic  psoriasis and this group includes: psoriasis- the prototype of psoriasiform dermatitis,1 and others as  seborrheic dermatitis,  pityriasis rubra pilaris (PRP), allergic dermatitis, atopic dermatitis , nummular dermatitis,  lichen simplex chronicus (LSC), prurigo nodularis,  pityriasis rosea (PR), inflammatory linear verrucous  epidermal nevus (ILVEN) and mycosis fungoides (MF). Besides, clinical features in one patient may differ at different times and the diagnosis get obscured.3 As there is varied clinical presentation, a definitive histopathological  diagnosis is essential in the treatment of such inflammatory dermatoses.1 It is a challenging  task even to experienced pathologists to give a precise diagnosis every time and one relies also on clinical correlation. However, a stepwise systematic histopathological approach enables one to reach at a specific diagnosis in most cases.2

The morphometry parameters such as length of rete ridges, the length of dermal papillae and the ratio of length/average width of rete redges are statistically significant in the differentiating  psoriasis from psoriasiform dermatitis and can be used in addition to routine histopathology.3 As the prototype of psoriasiform dermatitis, psoriasis is a hyperproliferative skin disorder with increased epidermal turnover rate and mitotic index, proliferation markers Ki-67 & Cyclin D1 immunostain of keratinocytes can be added as diagnostic tools  to differentiate from other non- psoriasiform dermatitis.4  Expression levels of pRb and p53 were found to be higher in the psoriasis group in a study,  compared with the normal epidermis.5  Psoriasis is an autoimmune skin disease and regarded to be T- cell mediated disorder. CD4+ T-cells are important in initiating and maintaining the pathogenic process of psoriasis but cross-primed CD8+ T-cells are the main effector cells. Mixtures of CD4+ T cells and CD8+ T-cells are found in papillary dermis and epidermis of psoriatic lesion.  Psoriasiform lesions on the other hand show a high proportion of CD4+ T-cells in dermis.  Immunophenotyping of T-cell infiltrate in the lesion can be done in differentiating the cases.6

 

History of Psoriasis

History of psoriasis begins in Ancient Greece, when psoriasis, leprosy, and other inflammatory skin disorders were believed to be the same condition and the Greeks termed the skin diseases as  psora, lepra and lichen. Psora referred to itch, and lepra from lopos & lepo (the epidermis & to scale respectively). Hippocrates (460-377 BC) used the word lopoi to describe the dry, scaly, disfiguring disorders.  The Old Testament also lumped together many cutaneous   disorders, including leprosy and psoriasis, by the biblical term tsaraat or zaraath. Lepers were considered divinely punished, and cruelty was imposed upon those who were suffered from psoriasis and leprosy alike. Roman medical writer Celsus (25BC-45AD) first described papulosquamous diseases, suggesting as psoriasis one of these. Galen (133-200) first used the term psoriasis, but his description was likely represented seborrheic dermatitis. Gilbert (1797-1866) and Hebra finally distinguished the clinical picture of psoriasis from that of leprosy.7

 Epidemiology

Although psoriasis occurs worldwide, it is a common chronic inflammatory skin disorders affecting 1.5-2% population in the western countries and 1.3% in general population.6 The prevalence of psoriasis is low in certain ethnic groups such as the Japanese.8

Psoriasis can present at any age and has been reported at birth and in older people of advanced age. A bimodal age of onset has been recognized in several large studies. The mean age of onset for the first presentation of psoriasis can range from 15 to 20 years of age, with a second peak occurring at 55–60 years. Henseler and Christophers studied a series of 2147 patients and also reported as two clinical presentations of psoriasis, type I and II. Type 1 begins on or before age 40 years; Type II begins after the age of 40 years. Type I disease accounts for more than 75% of cases. Patients with early onset, tended to have more relatives affected and more severe disease than patients who have type II psoriasis. In addition, strong associations have been reported with human leukocyte antigen (HLA)-Cw6 in patients with early onset of psoriasis. The course and progress of psoriasis is unpredictable. In one study, 39% of patients reported complete remission of disease at the age between one and 54 years. Higher figures have been reported in Japan.8 A family history of the disease is common. Approximately 30% of patients have a first-degree relative with psoriasis, and the risk of psoriasis increases with the number of affected relatives a patient has.9

 Lifestyle and Morbidity

There is a link between cigarette smoking and psoriasis severity. There is sufficient evidence that aggravation of psoriasis is associated with alcohol consumption. Various recent studies report association of diabetes and other cardiovascular diseases to the severe form of psoriasis.10 Recent research suggests that patients with psoriasis have a systemic inflamma­tory state, putting them at increased risk of cardiovascular complications, including metabolic syndrome, peripheral vascular disease, stroke, myocardial infarction, and cardiac death.11  Psoriasis is increasingly being recognized as a disease that not only affects the skin but also has multi-systemic implications. Increasing epidemiological evidence suggests that patients with psoriasis may be more obese compared with the general population. Although the exact mechanism underlying the epidemiological association between psoriasis and obesity is uncertain, researchers have theorized that adipocyte elaboration of pro-inflammatory cytokines may exacerbate psoriasis.12 Many, but not all, studies have shown a positive association between metabolic syndrome and cardiovascular disease (CVD) and psoriasis, especially for (young) patients with moderate to severe disease. A recent study showed that Glyc A, which is a novel biomarker for systemic inflammation  was associated with psoriasis.13  Nearly 60% of psoriasis patients and 40% of psoriatic arthritis patients report their disease as a large problem in their everyday life. Psychosocial limitations of both diseases include enduring low self-esteem, feeling physically unattractive or sexually undesirable and avoiding social activities.14

Etiology

Etiology of psoriasis remains unknown,  even though, it is believed to be multifactorial with numerous key components including genetic susceptibility, environmental triggers in combination with skin barrier disruption and immune dysfunction.15 Various data suggest that infection is an important trigger for psoriasis. Streptococcal throat infection can initiate and exacerbate chronic psoriasis.15 Another research has shown that the composition of the cutaneous microbiota is related to many dermatological diseases including  psoriasis, atopic dermatitis, and acne vulgaris.16

Genetic Factors

Epidemiological studies have demonstrated that psoriasis has an important genetic component, where the role of environmental triggers (e.g., stress, mechanical trauma and streptococcal infections) is well documented.  Familial recurrence is also documented and disease concordance is higher in monozygotic than dizygotic twins. So, psoriasis is widely regarded as a multifactorial disorder caused by the interaction between inherited susceptibility alleles and environmental risk factors.17 The molecular genetic basis of psoriasis is complex, however, there are evidence that multiple genes are involved. Seven major psoriasis susceptibility loci have been reported and   a major susceptibility locus for psoriasis is at 6p21, referred to as PSORS1 and is overrepresented in all populations tested.  An association between psoriasis and other loci has also been reported on chromosomes 1p (PSORS7), 1q (PSORS4), 3q (PSORS5), 4q (PSORS3), 17q (PSORS2), and 19p (PSORS6). The strength of associations between such genes and susceptibility to psoriasis, apart from PSORS1, is variable, may relate, in part, to heterogeneity among different populations.8

 Mechanism

Psoriasis is characterized by an abnormally excessive and rapid growth of the epidermal layer of the skin. Abnormal production of skin cells and an overabundance of skin cells result from the sequence of pathological events in psoriasis.  Skin cells are replaced every 7 days in psoriasis rather than the usual about 53 days.18 Exclusive cellular “responsibility” for the induc­tion and maintenance of psoriatic plaques has not been clearly defined. Increased proliferation of keratinocytes and endothelial cells in conjunction with APC/T cell/monocyte/macrophage inflammation leads to the distinct epidermal and vascular hyperplasia that is characteristic of lesional psoriatic skin.15  CD8+ T cells seem to be dominant in the epidermis whereas the CD4 + T cells predominant subset in the dermis.  Activated CD4+ T cells produce a variety of  cytokines  including interleukin-2 (IL-2), TNFα and γINF.  TNFα stimulating keratinocytes may produce  IL -8, a potent T- lymphocyte and neutrophil chemoattractant  and may involved in the formation of Munro microabscess. γINF is believed to play in important role in the initiation of psoriatic lesion.18 In individuals with a genetic predisposition, external stimuli such as trauma (known as Koebner phenomenon), infections, stress, drugs, and alcohol can all trigger an initial episode of psoriasis. This initial trigger activates the innate immune system (Figure 1). Complexes of the antimicrobial peptide LL-37 and host DNA/RNA, both released by keratinocytes after common epidermal damage, activate plasmacytoid dendritic cells (pDCs) to produce large amounts of type I IFNs (α/β).  Subsequently, type I IFNs trigger maturation and differentiation of dermal dendritic cells (dDCs), and these dDCs then stimulate autoreactive T cells. Thereby, psoriatic autoimmune T cells are biased to produce Th1 and Th17/Th22 cells and which induce TNFα,/INFγ and IL-17/IL-22 production respectively. These mediators act on keratinocytes, leading to the activation and proliferation. In addition, type I IFNs directly upregulate IL-22 receptor (IL-22R) on keratinocytes, increasing their responsiveness to IL-22, and inhibits terminal differentiation and induces hyperproliferation of keratinocytes leading to epidermal hyperplasia, resulting psoriasis.19  In psoriasis , along with epidermal changes there is markedly increased dermal vascularity. The dermal vascular changes have been evaluated and shown to be due to change from arterial-type vessels to venules. The vascular changes may also precede the epidermal alterations, as shown in some studies.20

Clinical Features

Psoriasis is a papulosquamous disease with variable morphology, distribution, severity and course. Papulosquamous diseases are characterized by scaling papules and plaques.8  There are five clinical subtypes of psoriasis: vulgaris (plaque), guttate, pustular, inverse, and erythrodermic. The most common variant of psoriasis is plaque psoriasis, which includes approximately 85%–90% of psoriatic patients.15 The lesions are typically distributed symmetrically on the extensor surfaces of elbows, knees, and lumbosacral area and scalp. Psoriasis may also develop at the site of trauma, known as Koebner’s phenomenon.  Guttate psoriasis has drop-shaped lesions.  Inverse psoriasis forms red patches in skin folds. Pustular psoriasis presents as small non-infectious pus-filled blisters. Erythrodermic psoriasis involves widespread inflammation and exfoliation of the skin over most of the body surface. Psoriatic nail disease affects fingernails more commonly than toenails with small pits in the nail plate.8

Clinical Diagnosis

Psoriasis is a clinical diagnosis, and a skin biopsy is usually not necessary for a classic presentation of the disease.9 Diagnosis of psoriasis is usually based on the appearance of the skin. The characteristic lesions are sharply demarcated, scaly, erythematous plaques, papules, or patches on skin that may be pruritic and/or painful. They may be ovoid, round or irregular and often distributed symmetrically.9 Auspitz sign, named after Heinrich Auspitz, where there is pinpoint bleeding on removal of scales from the lesions of psoriasis. The test by which Auspitz sign is elicited by light tangential scratching with the edge of glass slide is called as Grattage test can be used  as clinical tool.21

Microscopic Diagnosis

Histopathology is considered as a “gold standard” for the diagnosis of most dermatological conditions including psoriasis.21 Common histological features in  psoriasis vulgaris are  hyperkeratosis, parakeratosis,  psoriasiform hyperplasia (Figure 2),  Munro microabscess / spongioform pustule of Kogoj,   thin suprapapillary plate, hypogranulosis, dilated capillaries in papillary dermis and dermal inflammation.1  However, other than  Munro microabscess, spongioform pustule of Kogoj and dilated tortuous capillaries in papillary dermis,  the remaining features , such as  parakeratosis,  psoriasiform hyperplasia can be seen  also in chronic eczematous dermatitis, such as atopic dermatitis, nummular dermatitis or allergic contact dermatitis and appear to be “psoriasiform”.18  However, following a systematic approach one can reach at conclusive diagnosis.

 

Tirumalae described a detail  microscopic description for definitive diagnosis as follows.2

 Scanning magnification

Examination of microscopic sections under scanning magnification forms the first step. The presence of evenly elongated, thin rete ridges with equally long dermal papillae can seen in one condition i.e., psoriasis. All other diseases exhibit an uneven psoriasiform pattern, where rete ridges are of uneven of lengths and thickness with thick supra-papillary plates as in PRP (Figure 3).

 Stratum corneum

The normal basket-weave pattern is lost in most cases and it is common to find parakeratosis. Parakeratosis can be present in small mounds or may be broad and confluent.  Mounds of parakeratosis can be present throughout the epidermis in conditions like psoriasis and dermatophytosis, in seborrheic dermatitis confined to the infundibular ostia or seen in both locations in PRP.  The classic “checkerboard” pattern i.e., parakeratosis staggered in both vertical and horizontal fronts is seen in PRP. Confluent parakeratosis is seen in dermatophytosis, syphilis, pityriasis lichenoides chronica (PLC) and most importantly, in MF. A sharply defined thick zone of parakeratosis alternating with orthokeratosis and mild papillomatosis are features of ILVEN.

Epidermis

Diminished or absent granular layer is seen in classic psoriasis, but is not a constant feature. Significant amounts of plasma in the scale crust goes against a diagnosis of psoriasis, and  large amounts of plasma and a rapidly proliferating epidermis are hostile to fungi. The finding of spongiotic vesicles in a psoriasiform dermatitis are seen in allergic contact/nummular dermatitis and seborrheic dermatitis. Spongiotic psoriasiform pattern is also encountered in patch-stage of MF.

 Changes in Papillary Dermis

Psoriasis is characterized by thin supra-papillary plates and elongated dermal papillae with dilated, tortuous capillaries. The presence of extravasated erythrocytes is a sign of acuteness and is seen in eruptive psoriasis and PR. Vertical streaks of collagen with uneven psoriasiform acanthosis and compact orthokeratosis is diagnostic of LSC. However, changes of LSC can be superimposed on any chronic, itchy dermatitis including psoriasis. In contrast, thin haphazard, wiry bundles of collagen are seen in the papillary dermis in PLC and MF.

 Nature of Infiltrate

Most of the psoriasiform dermatoses are dominated by lymphocytes in the dermis. When eosinophils are present among them, it indicates allergic/contact dermatitis or a drug eruption.  However, eosinophils are not a pre-requisite for diagnosis of either of these conditions. Histiocytes and plasma cells in a psoriasiform dermatitis with scale crusts give hints for secondary syphilis. Superficial and deep infiltrates are seen in syphilis and lichen striatus (LS).  In LS, there is a psoriasiform lichenoid pattern with deep infiltrates of lymphocytes along the adnexae, especially around the eccrine glands.

 Morphometry

Morphometric analysis of histological features can give a quantitative dimension to histopathology. The results of morphometry parameters such as length of rete pegs, the length of dermal papillae and the ratio of length/average width of rete pegs  can help in achieving the accurate diagnoses of psoriasis and psoriasiform dermatitis. In one study using eye-piece micrometer with the light microscope without special software and found statistically significant results in differentiating psoriasis from psoriasiform dermatitis. The mean length of rete pegs was 1.74 times greater in psoriasis when compared to psoriasiform dermatitis. The ratio of average length and width of rete pegs was considered representative of shape of rete pegs. The higher ratio of length over average width of rete pegs confirms the histopathological finding of slender long rete pegs, which favors the diagnosis of psoriasis.

The dermal vascular changes have been evaluated in different studies by electron microscopy and shown to be due to change from arterial-type vessels to venules. The vascular changes may also precede the epidermal alterations, as shown in some studies. Morphometric parameters of papillary dermal vessels in psoriasis and to compare these with psoriasiform lesions can also be used. Morphometric evaluation of dermal vasculature in psoriasis has shown increased endothelial and luminal volume of vessels compared to control subjects. On light microscopic evaluation of CD34 immunostained sections, biopsies from psoriasis showed much greater microvascular staining in the papillary dermis in comparison to psoriasiform dermatitis.20

 Role of Immunohistochemistry

  Ki-67 and Cyclin D1 Immunostaining

As Psoriasis is a hyperproliferative skin disorder with increased epidermal turnover rate and mitotic index,  proliferation markers Ki-67 (Figure 4) and Cyclin D1 immunostaining  can be used to differentiate psoriasis from non-psoraisis psoriasiform dermatitis( NPPD ) (Figure 5). In a study Ki-67 and Cyclin D1 positive cells were counted per mm2 of suprabasal and total epidermal area by Sezer. et al. 4

Psoriatic lesions have been shown to reveal a higher Ki-67 index compared with normal appearing, non-lesional skin.  In psoriasis group, suprabasal Ki-67 positive cells were more than 75% of the total epidermal Ki-67 positive cells in the epidermis (including basal and suprabasal cell population) in psoriasis group, whereas this ratio was lower than 75% (cut-off value) in NPPD group. The suprabasal/total epidermal count ratio for Cyclin D1 immunostaining was higher in the psoriasis group compared with the NPPD group, but a cut-off value to distinguish between these two was not detected, suggesting that Ki-67 is a more sensitive marker than Cyclin D1 in terms of the presence of a cut-off value and could be a useful tool for dermatopathologists to differentiate psoriasis from other psoriasiform dermatitis.4

 pRb and p53 Immunostaining

Expression levels of pRb and p53 were found to be higher in the psoriasis group  in a study,  compared with the normal epidermis.  The phosphorylation of pRb is associated with cyclin D1, cyclin E and P16.  pRb phosphorylation in psoriasis is incresed by the activity of cyclin D1 and Cyclin E and showed a higher expression of pRb than that of normal epidermal expressions.  Total basal layer cell counts for p53 expression were found to be significantly higher in psorisis group compared with the normal group.5 It is postulated that increased expression of p53 in psoriatic skin is a physiological reaction indicating the attempt  to counteract the proliferation and to repair DNA errors, and is most often expressed as an increased number of mitoses.22

 CD4+ and CD8+ cell distribution

Psoriasis also known as a skin disorder with immunological factors playing an important role in its pathogenesis, and lymphocytic infiltrates, mainly CD4+ and CD8+ cells provide a major contribution in the initiation and maintenance of the lesion. Psoriasis is an autoimmune skin disease characterized by T-cell mediated hyperproliferation of keratinocytes. It is regarded as a T-cell mediated disorder, mainly CD4+ (helper/inducer) lymphocytes along with CD8+ (suppressor/cytotoxic) subsets are known to be associated. T-lymphocytes in psoriatic lesion are in an activated state with expression of HLA-DR and IL-2 receptor. Mixtures of CD4+ T-cells and CD 8+ T-cells are present in papillary dermis and epidermis of psoriatic lesion. T-cell also plays a role in the pathogenesis of psoriasiform lesions, and reported a high proportion of CD4+ T-cells in the dermis on immunohistochemical analysis.6

A comparative study was done by Rana. et al to see the infiltration of CD4+ and CD8+  cells in psoriasis and psoriasiform lesions. The inflammation in the epidermis was present in about half of psoriasis cases and predominant cells were polymorphs. In the upper dermis, inflammation was evident in all cases with predominant cells were lymphocytes. Inflammation in the lower (reticular) dermis was present with mostly lymphocytes.

In psoriasiform lesions the inflammation was present in the epidermis in only few cases showing mixtures of polymorphs and lymphocytes. Within the upper dermis, all cases showed inflammation with predominant of lymphocytes. Few cases showed inflammation in the lower dermis with lymphocytes as majority of the cells. Immunohistochemical staining was done to see the distribution of CD4+ and CD8+ cells.  the distribution of CD4 positive cells was seen in psoriasis patients.  In the epidermis of psoriasis cases only one fifth were positive for CD4+ cells. In the upper dermis, CD4+ cells were present in 92% cases.  In the lower dermis about half of cases were positive for CD4+ cells.

D 8+ positive cells on the other hand were seen in the epidermis in 40% cases.  In the upper dermis, all the cases showed CD8+ positive cells.  In the lower dermis, CD8+ positive cells were found in large number of cases.

An immunophenotyping of the inflammation of psoriasiform lesions showing, CD4+ cells as predominant cells in the upper dermis. In lower dermis area showed 16% cases having CD4+ cells.

Various studies have shown that CD4+ cells are less frequent in psoriatic epidermis. In epidermis CD8+ cells were seen in 40% cases, and 8% in psoriasiform lesions. The difference was statistically significant.  In the upper dermis, one study has shown the mixtures of CD4+ and CD8+ T-cells in psoriasis, whereas psoriasiform group showed that CD4+ cells in abundance.

The difference in amount and pattern of CD4+ and CD8+ T-cells in the various compartments is helpful in differentiating between psoriasis and psoriasiform lesions especially in cases with borderline morphology.6

 Conclusion

Psoriasis, the prototype of psoriasiform dermatitis, is a genetically determined, inflammatory, and proliferative disease of the skin characterized by dull red, sharply demarcated scaly plaques. The two clinical signs, Auspitzs sign and the Grattage test have been described as pathognomonic of psoriasis when biopsy is not done.  However, these signs are present only in untreated patients. Psoriasis has many different clinical differentials as other psoriasiform lesions. Besides, the same patient can present at different times with a different clinical presentation.  Since satisfactory management of the condition requires both symptomatic and specific therapy, it is essential to reach a definitive diagnosis. The recurrent nature and prognosis of psoriasis differs from that of psoriasiform dermatitis, thus, further highlighting the importance of reaching the correct diagnosis. Clinical features, considered alone, may not be reliable. Histopathology is regarded as a gold standard for the diagnosis of most dermatological conditions including psoriasis and constitutes definite hard evidence, which can be preserved for future review.  Though to give a precise diagnosis one relies on clinical correlation, histopathology is essential as it is possible to assign specific diagnosis in most cases with a logical and systematic histopathological approach.  In selected cases morphometry, Ki-67 & Cyklin D1, pRb and p53 immunostaining of keratinocytes can be added when needed. The pattern of T-cell infiltrate by immunophenotyping can give an additional diagnostic clue.

Acknowledgement

The authors are grateful to Faisal Kabir Taj for his help in preparing the figure 1.

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  21. Mehta S, Singal A, Bhattacharya SN. A  study of clinicohistopathological correlation in patients of  psoriasis and psoriasiform dermatitis. Indian J Dermatol Venereol Leprol 2009;75:100.
  22. Baran W, Szepietowski J. C, Szybejko-Machaj G. Expression of p53 protein in psoriasis. Acta Dermatoven APA 2005;14(3):79-83.
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Correlation of Ki-67 Proliferating Index with Histological Stage and Grade in Colorectal Carcinoma

Correlation of Ki-67 Proliferating Index with Histological Stage and Grade  in Colorectal  Carcinoma

 *Sultana S,1 Islam N,2 Kabir E,3 Akhter S,4 Paul R,5 Shirin A,6  Khan AA,7 Jahan N8

Abstract
Colorectal carcinoma is the most  common cancer of gastrointestinal tract. It is the 3rd most  commonly  diagnosed  cancer  and  the  3rd  leading  cause  of  cancer  death. The  growth  of  tumor  in  colorectal   carcinoma  is  highly  variable  and  its  histological grading and  staging  has  important  role  in  diagnosis, treatment  and  overall  prognosis. To observe the Ki-67 expression in colorectal carcinoma and find out the possible correlation of Ki-67 proliferating index with histological grading and Duke’s staging. This  cross sectional  study  was  conducted  at  Sir Salimullah Medical College  from  July 2014  to June 2016. 98  patients  with colorectal carcinoma  enrolled  in  this  study  by  purposive  sampling. The H&E staining was done on paraffin embedded tissue sample. Ki-67 expression by IHC method. Ki-67 is a proliferation associated  nuclear  antigen  which  can be recognize by  MIB-1 monoclonal antibody, correlate with histological staging and grading in  colorectal carcinoma. Then tumours were graded according to WHO grading criteria and pathological staging was done according to Duke’s staging system and immunohistochemical staining for Ki-67 antigen expression. Results were subjected to statistical analysis. The results were considered to be significant when the P< 0.05. Ki-67 proliferative index was high in well and moderately differentiated adenocarcinoma but low in poorly differentiated which is statistically significant (p <0.05).  Ki-67 expression was high in early Duke’s stage A and B but low expression  in advanced Duke’s stage C (p>0.05). The result of  this  study will enlighten the clinician regarding  the need for  doing Ki-67 in  colorectal carcinoma which   would  contribute to better understanding  of the  treatment as well as prognosis.

[Journal of Histopathology and Cytopathology, 2018 Jul; 2 (2):125-133]

Key words: Colorectal carcinoma, Immunohistochemistry, Ki-67

  1. *Dr. Sahela Sultana, Assistant Professor, Department of Pathology, Dr. Sirajul Islam Medical College, Dhaka. sultana.sahela83@gmail.com
  2. Professor Dr. Nasimul Islam, Professor and Head, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  3. Professor Dr. Enamul Kabir, Professor, Department of Pathology, Sir Salimullah Medical College, Dhaka.
  4. Salma Akhter, Assistant Professor, Department of Pathology, Universal Medical College, Dhaka.
  5. Rita Paul, Assistant Professor, Department of Pathology, Ibrahim Medical College, Dhaka.
  6. Afroz Shirin, Assistant Professor, Department of Pathology, Enam Medical College Savar, Dhaka.
  7. Abu Anis Khan, Assistant Professor, Department of Pathology, International Medical College, Dhaka.
  8. Nusrat Jahan, Lecturer, Department of Pathology, Sir Salimullah Medical College, Dhaka.

 

*For correspondence

Introduction

Colorectal carcinoma is the most common malignancy of gastrointesinal (GI) tract and is a major cause of morbidity and mortality worldwide.1 Colorectal cancer accounts for 10% of all cancers and it is the 2nd leading cause of death from malignancy in the industrialized world.2 There are nearly one million new cases of colorectal cancer diagnosed worldwide each year and half a million death.3 In 2013, there were an estimated 1,177,556 people living with colon and rectal cancer in the United States and the number of new cases of colon and rectal cancer was 41.0 per 100,000 men and women per year.4 Regarding age  incidence of colorectal carcinoma, recent reports show that in the USA it was the most frequent form of cancer among the person aged between 60-70 years and fewer than 20% of cases occurs before the age 50.5 The incidence of  colorectal cancer in Bangladesh is exactly not known but estimated population are approximately 15,10.1%.6 The distribution of colorectal carcinoma worldwide seems to be related to industrialization and socioeconomic standard and the incidence rate is higher in industrialized countries including Western Europe, Scandinavia and North America, whereas in the developing countries (sub-Saharan, Africa and Asia) the incidences appear to be lower.7

There are several staging system for colorectal carcinoma among these TNM and Duke’s staging systems are the most common way of staging and grading of colorectal carcinoma.8 The American Joint Committee (AJC) and the Union  for International  Cancer control (UICC) joined to produce the TNM system, which attempt to record clinical and pathological data, guide therapy and forecast prognosis, all in one.9 Whereas  the  classification   into  Duke’s stage A,B,C  cases   is  the  measurement  of  the  boundaries  reached and both   methods   permit the grouping  of  cases into favorable and unfavorable outcome.10 Histologically  the tumor is graded according to WHO grading criteria as well differentiated, moderately differentiated and poorly differentiated and the histological appearance of colorectal carcinoma may vary considerably with its major importance being related to prognosis.10

The use of monoclonal antibodies raised against specific antigens associated with the cell proliferation.11  Ki-67 is a proliferation associated nuclear antigen expressed in all cycling cell except resting cell in the G0 phase and it reflects cell in the S/G2+M phases in particularly.12  MIB-1 is a monoclonal antibody and it recognizes the Ki67 nuclear antigen in the formalin fixed paraffin embedded tissue section.13 Ki-67 expression is estimated as the percentage of tumors cells positively stained by the antibody with nuclear staining.12 The importance of Ki-67 as an indicator of tumor behavior and in colorectal cancer this index may be used as a marker of prognosis.12

The proliferative activity as measured by Ki-67 antibody is closely associated with histological  grade and stage.2 In 2008 Uzma Nabi, Nagi A H and Waqas Sami, Department of Pathology, University of Health Sciences, Lahore, Pakistan conducted a study on Ki-67 proliferating index and histological stage and grade  of colorectal carcinoma and observed that proliferative index is high in well and moderately differentiated  adenocarcinoma and in an early Duke’s stage (A or B).2 But Ki-67 proliferating activity is low in poorly differentiated tumour and  in an advanced Duke’s stage C.13,8 But there are some studies of Lanza, Cavazzinil  in 1990 and  Yokoyama N, Okomoto H in Japan in 2005 contradicting the above mentioned  association of Ki-67 versus grading and staging of colorectal carcinoma, they concluded that proliferating index of Ki-67 was increasing with increasing grade, stage.14, 15

Methods

This  cross sectional study was conducted among the 98 histopathologically diagnosed patients having colorectal carcinoma  over a period of two years in the department of surgery, Sir Salimullah Medical College. Study population were the patients having colorectal cancer underwent surgical  treatment in the department of surgery of Sir Salimullah Medical College. The proliferative activity as measured by Ki-67 antibody is closely associated with histological  grading  and staging of colorectal carcinoma. The representative sections were submitted for Immunohistochemical staining. The Ki-67 immunostaining were performed according to manufacture’s recommendation, using the MIB-1 clone (DAKO, Carpenteria, CA & Ventena Medical System, Tucson, AZ). Ki-67 immunostainined slides were examined via light microsccopy. Positive Ki-67 staining was observed brown granular nuclear staning. For Ki-67 scoring the most positive area of the tumor was selected avoiding foci of inflammation. The number of positive nuclei were counted in 500 tumor cells in a high power field. The average of the counts over the same slides was taken and expressed as the percentage of Ki-67 positive cells  in the tumor.

Statistical analysis were performed in SPSS statistical software program, Version 17.0. To correlate histological grading and staging of colorectal carcinoma with Ki-67 proliferating index were performed with  Mann- Whitney U test. The result were consider to be  significant when P<0.05. One way ANOVA followed by Bonferroni test was performed to compare between groups.

Results

98 cases were included in the present study.  Age incidence ranged from 28-78 years and their mean ± SD 47.38 ± 10.37. Maximum patients (30.6 %) were found in 41-50 years age group where M: F was 1.72: 1 (Table I). Regarding site of tumor more than 50% of patient had tumors in the left side of colon and in the rest of the cases tumors were present in caecum (26.5%; Table-II).  98 cases having different sizes of tumors and in most of the cases fifty nine cases (60.2%) tumors size were 3-4 cm and  their mean ± SD 4.8 ± 1.8 (Table-III). Different morphological types of tumor were observed in present study. In maximum forty cases (40.8%) the morphological types of tumors were ulcerative and only five cases (5.3%) tumors were infiltrative type. Rests were annular (33.5%) and polypoid (20.3%; Table IV).

In the present study, tumors were graded according to WHO grading system into well differentiated, moderately differentiated, poorly differentiated and grouped into A, B, C accordingly. The maximum cases 69 (70.4%) of colorectal carcinoma were well differentiated and their mean Ki-67 proliferating index was 47.83 ± 15.23. There were significant differences among the groups (A vs B vs C), when mean proliferating index of Ki-67 were compared among the three groups. The result was found statistically significant (P<0.05). However when compared in between groups only A vs C (between well differentiate & poorly differentiate) groups were found also statistically significant (P<0.05). In the present study, it was also observed that with increasing grade, Ki-67 proliferating index decreases (Table V) .

Regarding staging of the tumor (Duke’s staging) where  maximum  cases (44.8%) were in stage B1 and their mean Ki-67 proliferating index was 46.25±14.06. There was no significant differences among the stages and the result was not statistically significant (P>0.05). In the present study it was also observed that with increasing stage of the tumors, there was decreasing Ki-67 proliferating index (Table VI).

Table I: Distribution of patients according to age group with male female ratio (n=98)

Age groups         Frequency            M: F      Percentage
Total  Male Female
≤30

 

 10    7    3           2.3:1       10.2
31-40 23   13   10           1.3: 1       23.5
 

41-50

  

30

  

19

  

11

  

1.72: 1

  

30.6
 

51-60

  

26

  

17

  

9

  

1.88: 1

  

26.5
 

≥60

  

9

  

9

  

0

  

9.00: 0

  

9.2
 

Total

  

98
 

Mean ± SD

 

  

47.01± 10.99
Range (Min-Max)                                              28 – 78

 

 

Table II: Distribution of tumors according to site (n=98)

Site of tumor Frequency (%)
Left side of colon
Sigmoid colon 34 (34.7)
Transverse colon 24 (24.6)
Rectum 14 (14.7)
Right side of colon
Caecum 26 (26.5)
Total 98 (100)

Table III: Distribution of patients according to tumor size (n=98)

Tumor size (cm) Frequency (%)
1 – 2 14 (14.3)
3 – 4 59 (60.2)
5 – 6 19 (19.4)
Mean ± SD 4.8 ± 1.8
Total 98 (100)

 

Table IV: Distribution of patients according to morphological types of tumor (n=98)

Morphology Frequency (%)
Ulcerative 40 (40.8)
Annular 33 (33.5)
Polypoid 20 (20.3)
Infiltrating 5 (5.3)
Total 98 (100)

 

Table V: Relation of Ki-67 proliferating index with histological grading (n=98)

Grading Frequency

n(%)

 Ki-67 expression

(Mean ± SD)

 P
Well differentiated (A) 69 (70.4) 47.83± 15.23
Moderate differentiate
(B)		 15(15.3) 46.33 ± 18.07
Poor differentiated (C)  14(14.3) 35.35 ± 11.17
Statistical analysis
A vs B vs C 0.023*
A vs B 1.000ns
A vs C 0.019*
B vs C 0.162 ns

ANOVA followed by Bonferroni test was performed to compare between groups

Table VI: Relation of Ki-67 proliferating index with histological stage (n=98)

 

Duke’s staging

 

 Frequency

n (%)

 Ki-67 expression

(Mean ± SD)

 p
Stage A 3 (3.3%) 53.33 ± 20.81 0.727ns
Stage B1 44 (44.8) 46.25 ± 14.06
Stage B2 20(20.5) 43.25 ± 14.06
Stage C1 14(14.8) 44.64 ± 21.07
Stage C2 17(17.5) 42.05 ± 13.69

 

ANOVA test was done to measure the level of significance.

 

 

 

 

 

Figure 1. Photomicrograph of histopathological section of well differentiated adenocarcinoma of colon (H&E method x100)

 

 

 

 

 

Figure 2. Photomicrograph of   well differentiated adenocarcinoma stained with Ki-67 immunostain showing high proliferative Index(x100).

 

 

 

 

Figure 3. Photomicrograph of histopathological section of poorly differentiated adenocarcinoma of colon ( H& E method x400).

 

 

 

 

 

Figure 4. Photomicrograph of   poorly differentiated adenocarcinoma stained with Ki-67 immunostain showing low Proliferative Index (x400).

Discussion

Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide.16 The incidence of cancer colon and rectum was 41.0 per 100,000 men and women per year and the number of deaths was 15.1 per 100,000 men and women per year in Bangladesh.17 Management of  colorectal carcinoma depends on a number of  morphological and biological factors which include the pathological tumor stage (including involvement of lymph nodes, breach of serosa, distant spread etc.), primary tumor characteristics (including depth of tumor penetration in the bowel wall, histological subtype, histological grade and differentiation, venous and lymphatic invasion, perineural invasion and lymphocytic infiltration), status of surgical resection margins (free or involved).18 With assessment of tumor cell proliferation may predict tumor behavior.19 The aim of this study was to evaluate the proliferating index (PI) in formalin fixed, paraffin embedded tissue section of colorectal carcinoma, using monoclonal MIB-1 antibody (Ki-67) and to assess the relationship between proliferative index (PI) and various pathological findings in colorectal carcinoma including histological grade, and stage.

In the present study, the mean age of the patients was 47.01± 10.99 years and the highest number of malignant cases were seen in the 4th and 5th decades. Male female ratio was 1.72:1 and 1.88:1 in 4th and 5th decades accordingly. So, in present study, male were predominant than female. These findings were similar to other studies.20, 21

In 34.7% patients the tumors were located in sigmoid colon followed by caecum (26.5%), transverse colon (24.5%) and rectum (14.3%).  As per gross morphological type of cancer, maximum 40.8% were ulcerative and 33.5% were annular type and rest were polypoid (20.3%) and infiltrating type (5.3%). In one study, colon was more commonly affected site compared to rectum and most of the lesions were ulcerative.22 In another study rectum was the most common affected site and predominant lesions were annular.23

In present study, 60.2% of malignant cases had tumors size between 3-4 cm and 19.4% malignant cases had tumors size 5-6 cm. In a study in 2011 by Kornprat showed that maximum size of the tumor in colon cancer were in between 4.5 to 6.5 cm in majority of cases.24 In 2013 ASCO annual meeting showed a report where a study conducted on tumor size in colorectal cancer found that in majority of the cases the tumors sizes were in between 4-6 cm in colorectal carcinoma.25

In this present study it was observed that  the Ki-67 labelling index was high in grade I and grade II  compared to grade III. These results showed that proliferating index was low in poorly differentiated tumor compared to well and moderately differentiated tumor. When Ki-67 proliferating index was compared among three groups a statistically significant correlation was found in present study (p<0.05). These findings are similar to studies in Japan2 Pakistan12 and Finland.26 On the other hand, some of the studies showed contradictory result that Ki-67 proliferating index increased with increasing histological grade.26,27

In the present study, it was observed that Ki 67 labeling index was high in Duke’s stage A and B  and tumor in advanced stage (Duke’s C) have a low proliferating index  compared to tumors in  an early invasive stage (Duke’s A and B). Regarding the correlation of Ki-67 LI with staging, no statistically significant correlation was found. This result was similar with other studies in which they concluded that Ki-67 proliferating index was significantly lower in carcinoma in subserosa or deeper invasion compared to carcinoma with submucosa or muscularis mucosa invasion.2,8,26 In another study  contradictory results have been reported on associations of Ki-67 with prognosis and survival of colorectal tumors with a low proliferation index in Duke’s A and B tumors to be associated with survival impairment compared to those with high values.28 Tumors with high proliferative activity are known to be most responsive to radiotherapy and Willett and collaborators showed that radiation eradicates preferentially rapidly dividing cells in rectal cancer, whereas populations with slow proliferation show greater radioresistance.29

Conclusion

It is concluded that Ki-67 labeling index is high in well to moderately differentiated adenocarcinomas in an early Duke’s stage A or B compared to poorly differentiated adenocarcinomas, and  in an advanced Duke’s stage C. Thus Ki-67 proliferating index can be useful in a patient with colorectal carcinoma as a ancillary diagnostic support. Moreover, it may help in the prognostic evaluation of patient, survival as well as in considering them for post surgical treatment.

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