Clinical and pathological aspects of condyloma acuminatum – review of literature and case presentation

Abstract

Condyloma acuminatum (CA) is a pathology caused by the human papillomavirus (HPV). It is manifested by the appearance of warts in the vulvar, pubic, and anorectal regions, but can occur in other areas. It is a common disease that can be prevented by using measures such as condoms or vaccine. Topical, local, pharmacological, surgical, and excisional therapy options are available for this pathology. Macroscopically, it appears as a vegetative tumor, with a single implantation base that branches towards the periphery, with a cauliflower appearance. CA is defined microscopically by acanthosis, parakeratosis, papillomatosis and koilocytosis. Immunohistochemical studies can detect the presence of various HPV strains or viral antigens and can emphasize certain specific characteristics; e.g., in the case presented in this study, we observed that the tumor had a fulminant evolution due to a strong vascular base identified with anti-cluster of differentiation (CD) 34 antibody, by the existence of epithelial cells with a high degree of cell proliferation, as evidenced by the anti-Ki67 antibody, the inactivation of the tumor suppressor gene and the appearance of immunolabeling for the anti-p53 antibody, by the strong immunoreactivity for p63 which reveals the existence of cells with dysplastic and neoplastic transformation potential, but also by detecting the immunolabeling for p16^INK4a that is associated with the existence of HPV. Also, the tumor was immunoreactive for cytokeratin (CK) AE1/AE3, partially immunoreactive for CK5/6 in the basal layer and negative for CK7, which demonstrates the squamous epithelial origin of the described tumor. Subepithelial cells of the inflammatory system have been identified, such as macrophages immunolabeled with anti-CD68 antibody, T-lymphocytes immunolabeled with anti-CD3 antibody and rare B-lymphocytes immunolabeled with anti-CD20 antibody, which demonstrates the strong cellular response to remove the virus from the structure. Surgical and excisional treatment was helpful for the patient, because she was able to resume normal sexual activity and defecation, and on the other hand, microscopic studies showed the potential for malignant transformation of CA.

⧉ Introduction

Condyloma acuminatum (CA) is a predominantly sexually transmitted disease (STD) that is characterized by the appearance of fleshy papules and skin-colored in the anorectal region and is caused by infection with several strains of human papillomavirus (HPV). These formations are also called venereal warts, anal warts or anogenital warts [1].

In 1907, CA was classified as a cutaneous manifestation of syphilis or gonorrhea, but other authors used cell-free transmission experiments to demonstrate the virus’ infectious nature, injecting wart extracts into previously uninfected skin and observing the appearance of papillomatous eruptions in those locations [2].

Subsequent experiments and studies have shown that genital warts are benign proliferations of anogenital mucosa, caused by a viral HPV infection, recently detected by molecular biology studies [3].

Aim

The aims of this study were to emphasize the clinical and morphopathological characteristics of CA as described in the literature and to highlight the serious importance of prevention and effective therapy.

⧉ Epidemiology and risk factors

In the United States (US), genital HPV infections affect roughly 10–20% of the population, representing about 20 million people [4,5], whereas clinical signs occur in 1% of the infected population.

Since 1975, the incidence of this disease has been increasing in the US, with onset ages ranging from 17 to 33 years in 80% of cases [6]. The use of the medication can cause the condylomas to disappear, but the cause cannot be determined, thus the likelihood of recurrence is considerable (50–73%) [4]. Warts, on the other hand, can regress spontaneously without therapy [6].

According to some research, the virus can survive in the human body for the rest of one’s life, but other studies based on sensitive deoxyribonucleic acid (DNA) techniques have revealed that the patient’s immune system can respond in such a way that the virus is removed or suppressed at a level that cannot be detected by polymerase chain reaction (PCR) tests. The PCR tests also detected subclinical stages with a prevalence of 10% [6].

CA is one of the most frequent types of STD that can affect the general population [7]. In the US, between 500 000 and 1 000 000 new cases with clinical manifestation are detected each year [8,9]. In 2004, the cost of caring for patients with HPV, CA, or invasive cervical cancer was assessed to be around four billion dollars [10].

This phenomenon of increasing CA cases is inversely related to the age of onset of sexual activity and highly related to the number of sexual partners, with roughly half of new infections occurring in young adults aged 15 to 24 years [3].

HPV is a highly contagious virus that is spread primarily by sexual, anal, and genital contact, but also through vertical transmission or autoinoculation in rare circumstances [11]. As a result of unprotected sexual contact with a person infected with HPV, there is a risk of transmission of approximately 75%, increasing the risk of developing lifelong CA by 50% in sexually active individuals.

CA-associated risk factors are unprotected sex, STDs, immunosuppression, especially in patients with human immunodeficiency virus (HIV) and use of oral contraceptives or smoking [12,13].

Several studies have demonstrated that the use of condoms can help to reduce viral transmission [14]. Other research has found that condoms are ineffective at preventing viral transmission of strains that cause CA, but they may be more beneficial for men than for women [15].

Because the viral strains that cause CA are extremely contagious, 3/4 of unaffected people who have a partner who is infected develop this pathology within eight months of being infected [15]. Additionally, the presence of warts indicates the degree of infectivity, so the correlation between the presence of warts and the transmission is directly proportional [14]. Another mode of transmission is direct, which occurs during birth if the mother has warts in the vaginal area, or self-inoculation, which happens when the warts are touched with other parts of the body, particularly the hands [15].

Studies have reported that the virus can be transmitted through the sharing of swimwear, underwear, or non-sexual touch during routine care, such as changing the baby’s diaper. Children’s warts are more likely to be caused by cutaneous HPV types than by subtypes 6 and 11, and the presence of CA in a child’s genital anal area may be a sign of sexual abuse [16].

⧉ Latency and recurrence

Over 90% of infected cases heal spontaneously due to the immune system intervention. Infected cells acquire a dormant state and can reappear at months or even years’ intervals [17]. During this latency period, the virus can be transmitted to the sexual partner in 70% of instances. Recurrence of warts in patients with a history of HPV infection can also be caused by reinfection with this virus or a recurrence of a prior infection. According to studies, around a third of cases are recurrent [8].

⧉ Virology, dermatopathology and pathophysiology

HPV infection begins with virus inoculation into the epithelial structure via microabrasions. HPV is a part of the Papovaviridae family of non-enveloped, double-stranded DNA (dsDNA) viruses [18]. Although the virus is capable of penetrating both the mucosal epithelium and the corium of infected tissue, viral replication occurs in the epithelial basal layer, in the structure of cells with tropism, called keratinocytes. The mucosa that is susceptible to HPV infection can be found in the vagina, the cervix, but also in the perianal regions of women and men, in the penile and periurethral areas of men. Viral replication results in the formation of warty papules or warty plaques. HPV’s viral genome has six early-open reading frames (E1, E2, E4, E5, E6, E7), as well as two late-open reading frames (L1, L2). The first ones play a role in regulating and coding of proteins involved in viral replication and determine cell transformation. Furthermore, they are responsible for encoding the proteins contained within the viral capsid [18].

There may be more L1 models that define distinct DNA-virus replication patterns, classifying the virus into additional subtypes. High-risk (HR) HPV strains directly integrate its genetic material into the host cell, resulting in uncontrolled activation of E6 and E7 genes and transcription of oncoproteins and inactivation of p53 and retinoblastoma (Rb) tumor suppressor genes, but also cellular proliferation and subsequent evolution toward tumorigenesis (Table 1) [19,20,21].

Table 1.

Comparison between LR HPV subtypes versus HR HPV subtypes

	LR HPV subtypes 6 and 11	HR HPV subtypes 16 and 18
Clinical implications	75–90% cases of CA, respiratory papillomatosis	70% cases of all dysplastic lesions, Bowen’s disease, and invasive cervix cancer
Pathophysiological mechanisms	Viral DNA undergoes independent viral replication	Viral DNA integrates into the host genome and causes inactivation of tumor suppressor genes
Connections with different types of verrucous carcinoma	Oral florid papillomatosis, Buschke–Löwenstein tumor	Oral florid papillomatosis

CA: Condyloma acuminatum; DNA: Deoxyribonucleic acid; HPV: Human papillomavirus; HR: High-risk; LR: Low-risk

HPV integration into host cells causes cell infection and the appearance of keratinocytes with altered and atypical morphology, called koilocytes. They contain small nuclei that are eccentrically positioned and the cells having an increased volume. Additionally, a characteristic halo appears perinuclear. Increased epithelial cell volume has a pronounced acanthosis, with the appearance of papillomas, parakeratosis, and hyperkeratosis, as well as the loss of the granular cell layer. The dermis is highly vascularized, with thrombosed capillaries. Moreover, infected basal cells migrate to the adjacent layers, amplifying the viral gene. The assembly of virions takes place in the superficial layer of the epithelium, from which they are eliminated to infect their own or foreign adjacent tissues. CA has an exophytic phenotype because of viral effects on epithelial structure [22].

⧉ Clinical presentation

CA does not often cause serious damage to patients, but more severe cases might emerge, obstructing and interrupting the normal functionality of certain anatomical parts due to the increased size of warty tumors. Regarding HPV infection, an incubation period of three to eight months may occur until the onset of clinical manifestations. Clinical signs often manifest 2–3 months following interaction with the source of infection [23]. Occasionally, the virus remains latent, persisting in the epithelial tissue for an extended amount of time without manifesting clinically. According to studies, up to 40% of cases remain clinically undetected, the virus can be confirmed using DNA tests performed with samples taken from suspected virus-infected locations [24].

Managing a case of CA can also have several implications, involving screening examinations for STDs and HIV, as well as counseling on how to avoid acquiring or spreading STDs, HPV or HIV. Moreover, certain investigations should be performed to detect possible immunosuppression. The patient might be directed into cigarette cessation programs, if necessary. After the onset of clinical manifestations, CA may develop in several regions and increase in size or may regress spontaneously. Around 30% of CA cases spontaneously regress approximately four months after onset. Unfortunately, long-term remission is not always attainable, and most CA reappears three months after infection, even with adequate therapy [25]. The persistence of this infection is facilitated not only by host immunosuppression, but also by HR HPV strains, and viral infection suppression is directly related to the presence of CD4+ cells in the epithelial structure, which play a role in local immune regulation [25,26].

Condylomatous lesions are most frequently found on moist mucous membranes of the anogenital or oral regions following oral sexual contact with an infected partner [9]. They can have a variety of features, such as flat, cauliflower or peduncled form [26]. They can develop individually as solitary keratotic papules or plaques, although they are most commonly seen agglutinated. The clinical presentation begins with flesh-colored papules on the skin measuring 1–2 mm in diameter, followed by a growth in volume to a few centimeters or even a giant shape. The expansion of these lesions is related to the appearance of pain during sexual intercourse or with the hinder of eutocic birth. The warty appearance of the CA may be variable: it may be white, pink, purplish to reddish-brown colored, flat, warty or cerebriform. Symptoms that may occur are severe discomfort, itching, burning, bleeding on contact with clothing or during intercourse, difficulty urinating or defecating. In men, the lesions appear around the penis or even intraurethral and may be associated with dysuria, hematuria, or active bleeding. Scrotal lesions are extremely rare in immunocompetent organisms. Several patients described an unpleasant smell of secretion from the affected areas [11, 27].

⧉ Complications of untreated HPV infection

Low-risk (LR) HPV strains (6 and 11), but even HR HPV strains (particularly 16 and 18), can cause verrucous carcinoma (VC) with a low degree of differentiation (G3) [28]. VC is classified clinically and pathologically into numerous subtypes based on its anatomical location. If they manifest in the oral cavity, they are called oral florid papillomatosis; if they manifest in the anogenital region, they are called giant condyloma of Buschke and Löwenstein, and if they appear in the palmo-plantar region, they are called carcinoma cuniculatum. All these tumor subtypes are invasive locally but rarely metastasize [29].

⧉ Positive and differential diagnosis

CA may be detected with a thorough clinical history and physical examination. The 3–5% Acetic Acid test may be effective in the subclinical phases for identifying warty lesions that become white in infected areas [30]. The use of anoscopy or colposcopy can aid in the diagnosis of warty lesions [31].

Biopsy in the suspected region is necessary to obtain the correct diagnosis only when lesions with suspected malignancy or an increased risk of malignant transformation are present and are associated with ulceration, immobility, a sudden change in the appearance of the lesion or non-response to treatment [27]. Biopsy is performed to detect squamous cell carcinomas (SCCs), such as VC, Bowenoid papulosis (BP), and other similar lesions, but is frequently not required for diagnosis.

Differential diagnosis of CA includes the following entities: molluscum contagiosum, condyloma latum (CL), vulvar papillomatosis, pearly penile papules, angiokeratomas of the scrotum or vulva, skin tags, SCC, BP, seborrheic keratosis [32,33,34,35].

Molluscum contagiosum is characterized by the presence of papules that are normally asymptomatic but have a smoother surface than CA and are shaped like a dome, with a central umbilication that may be highlighted by applying a small amount of liquid nitrogen. CL is a manifestation of secondary syphilis that commonly appears as wet, sometimes weepy plaques in the anogenital area. Clinically, patients should be evaluated for further syphilis signs and symptoms. If accessible, darkfield microscopy can be used to visualize spirochetes to differentiate CL from CA. Vulvar papillomatosis is a typical female pathology distinguished by smooth papillary projections that each originate from a distinct base near the vaginal introitus. By contrast, numerous papillary projections can emerge from a single base in CA. Pearly papules penile develop along coronal sulcus and the penile corona in uncircumcised men and can be clinically distinguished from CA by their monomorphic shape and characteristic distribution. Another disease to consider is angiokeratomas of the scrotum or vulva. In contrast to CA, these papules have a distinctive vascular (red or purple) appearance. The skin tag smooth surface and pedunculated appearance usually separate them from CA. SCC is a disease that should be regarded when lesions ulcerate or do not react to commonly effective therapies for CA, or in immunocompromised individuals, or those with atypical lesions. Biopsy is indicated in those instances, as described above. BP develops because of HPV 16 infection and has a high probability of progressing to invasive SCC. It presents clinically as flat, but hyperpigmented papules, in contrast to CA. Histopathologically, seborrheic keratosis lacks koilocytes, unlike CA. Giant CA (Buschke–Löwenstein tumor) is a type of VC caused by HPV 6 infection that typically affects the glans penis and appears considerably less frequently on the perianal or vulvar tissue [32,33,34,35].

⧉ Therapy

Currently available treatments for CA are primarily focused on removing wart growth rather than eradicating the underlying viral infection. There is limited evidence that current therapies are effective at eradicating long-term genital warts or if they play a significant role in preventing warts from developing into potentially malignant lesions. Currently, a variety of therapies are used, each with a distinct cost, side effect profile, overall effectiveness, and duration of treatment. To date, no therapy has been identified as the best standard of care for CA; alternatively, therapy is chosen according to the particularities of the patient [36]. CA is treated in three main ways: locally [Podophyllotoxin, Imiquimod 5% cream, Imiquimod 3.75% cream, Sinecatechins 15% ointment, Podophyllin, 5-Fluorouracil (5-FU)], surgical and destructive [Trichloroacetic Acid (TCA), cryotherapy, electrosurgery, scissor excision, carbon dioxide (CO₂) laser], and systemic [Interferon (IFN)] [37].

Podophyllotoxin

Podophyllotoxin is recommended as 0.05% solution or gel and 0.15% cream (Grade A). Podophyllotoxin is a purified extract from the Podophyllum plant that can bind to cellular microtubules, stop mitotic division, and induce wart necrosis within 3–5 days of topical application. As the wart’s necrosis, the resulting minor erosions recover within a few days. This therapy is regarded safe and successful even if it is administered by the patient; it is available as a solution, cream or gel and must be administered twice daily, three consecutive days a week, for a maximum of four weeks. Typically, a cream or gel is used to treat vaginal or anal lesions, and a solution is utilized to treat penile lesions [37,38]. Podophyllotoxin has a success rate of 45–77%, but a recurrence rate of around 38%, according to studies. Warts which do not heal after four cycles of therapy should be considered for alternative treatment option [39,40,41].

Imiquimod

Imiquimod 5% cream (Grade A) is a topical immunomodulatory agent that can be applied by the patient, and it was firstly used in 1997 to treat external CA. Although its mechanism of action is not clearly known, Imiquimod is thought to activate immune cells by binding to membranous Toll-like receptors [42,43]. These changes result in the release of several cytokines such as tumor necrosis factor-alpha (TNF-α), IFN-α and interleukin-6 (IL-6) factor, which generate an inflammatory response that promotes wart removal. In addition, a decrease in messenger ribonucleic acid (mRNA) expression was observed for CK proliferation markers but also in viral load measured by HPV DNA, in patients treated with Imiquimod [44,45,46]. For the treatment of CA, Imiquimod is applied three times a week for up to 16 weeks. Although multiple clinical trials have validated the efficacy of Imiquimod 5% cream treatment for CA treatment, but the prolonged length of treatment may impair patient compliance [47].

Imiquimod 3.75% cream (Grade A) was recently approved by the Food and Drug Administration (FDA) for the treatment of CA. Although the cure rate is lower than Imiquimod 5% it is considered that due to the shorter duration of use, patients’ compliance is higher. It is applied daily for a maximum of eight weeks. Local side effects are milder than those associated with Imiquimod 5%, and no systemic adverse effects has been reported [48,49].

Sinecatechins

Sinecatechins 15% ointment (Grade A) is a botanical extract approved by the FDA in 2006 for the treatment of genital warts. The active ingredient is a green tea extract that contains Sinecatechins, which are believed to have antineoplastic, antioxidant and antiviral properties. Although the mechanism of action is unclear, it is believed that it modulates the inflammatory response by inhibiting the activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) transcription factors. Sinecatechins 15% cream is applied locally on lesions three times a day for up to four weeks. If no improvement is noticed within the first few weeks of treatment, a more intensive kind of treatment is used [50,51].

Trichloroacetic Acid

TCA 80–90% solution (Grade B) is a destructive chemical that cauterizes, oxidizes, and erodes mucous membranes and skin. Generally, is prepared in solutions at a concentration of 80–90% and must be administered by a doctor. Successful treatment of CA with TCA usually requires more applications, healing has rarely occurred after a single application. TCA is a cheap, cost-effective therapeutic option that requires continuous administration. The product’s destructive capacity typically extends beyond the superficial wart to the underlying viral infection, with cure rates ranging from 70% to 80% and a recurrence rate of roughly 36% [52,53]. In an obstetric trial that evaluated TCA 85% solution used to treat 50 women with external genital warts, all lesions were eradicated after two to five months of treatment. In the first six months of follow-up, there was no recurrence in the group of patients studied, but in the second follow-up period of six months, nine patients presented with recurrent warts. TCA is a suggested treatment option for CA because it is associated with a low rate of morbidity and a high rate of therapeutic success [54].

Cryotherapy

Cryotherapy (Grade B) is a technique that involves destruction of the affected tissue with a freezing agent such as liquid nitrogen or nitrogen oxide. Temperatures must be extremely cold to produce permanent vascular and skin damage. This results in an inflammatory response and necrosis, followed by the elimination of the damaged cells. Cryotherapy is often effective when small and multiple lesions in the vulva or penis are treated. Cryotherapy is widely regarded as a very effective and cost-efficient therapy, with an injury elimination rate between 79% and 88% seen within the first three months of treatment; this indicates a more beneficial outcome than TCA [55]. Several limiting factors, such as contact time and temperature of use, can affect the efficacy of this therapy. Local ulcers, potentially permanent scars, infections, painful blisters, and skin depigmentation are all common side effects of cryotherapy, which can be more severe than with TCA. Cryotherapy has local effects and is generally the treatment for pregnant women with numerous warts [55].

Electrosurgery

Electrosurgery (Grade B) is a method that burns and destroys warts using high frequency electric currents in the form of electrocautery or thermal coagulation. Curettage is used to remove the damaged tissue. This method is especially helpful for treating smaller warts on the vulva, penis, or rectum. These procedures should not be used on large lesions because they cause permanent scars. Electrosurgery is a highly effective procedure, with a success rate of approximately 94% six weeks after treatment. These results demonstrate that electrosurgery is comparable to cryotherapy in terms of long-term efficacy. Electrosurgery is a rather painful procedure that requires local or general anesthesia; often, post-procedural soreness is the only adverse effect [56,57].

Surgical scissor excision

Surgical scissor excision (Grade B) is one of the oldest methods of treating CA and has long been considered the first line of treatment. This involves the physical removal of genital warts with a scalpel or scissors and suturing healthy skin. It is associated with a success rate of up to 74%, one year after the intervention. Although this procedure is regarded outdated nowadays, it is appropriate for huge warts that do not respond to conventional treatment or cause obstructions; examples include warts affecting the urinary meatus. Additionally, surgical excision remains the primary treatment option for neoplastic tumors suspected of malignant transformation that must be followed-up histopathologically. Mohs surgery is a more advanced surgical procedure for genital wart removal. Although this procedure is most commonly used to remove carcinomas, Mohs surgery consist of removing the skin in very thin layers and immediately analyzing it microscopically for abnormal features. Further slices of skin are removed in the presence of viral cell features, until healthy tissue is reached. The obvious advantage of this procedure is that it preserves the maximum amount of healthy skin and results in the appearance of minimal scars [58].

CO₂ laser therapy

CO₂ laser therapy (Grade B) is based on the application of a concentrated infrared light beam to the target tissue, which heats and finally vaporizes it. Light’s intense energy has the property to cauterize the vessels, ensuring a bleeding-free procedure. The laser beam’s spatial ‘concentration’ enables accurate tissue excision, resulting in a very small scar and complete healing. The efficacy of CO₂ therapy in treating CA remains debatable. Laser therapy has a success rate of between 23% and 52% and is regarded less successful than other surgical procedures. Rates of recurrence are increased up to 77%. Side effects are generally limited to burning tissue in the vicinity of the lesion. Despite these apparent disadvantages, the laser has a substantial effect, allowing for a more thorough and effective viral treatment than is possible with other methods of surgical treatment. This approach is still preferable for pregnant women with big warts that are resistant to cryotherapy and for immunosuppressed persons [59].

Therapies not generally recommended

Due to its low efficacy and toxicity, the routine use of 5-FU, Podophyllin or IFN is not recommended for primary care [60].

⧉ Prevention

Numerous preventive strategies can be applied to avoid HPV infection and the development of CA, including the use of condoms, however they do not give 100% protection, and there may be skin contact in other locations during sexual intercourse. Also, limiting the number of sexual partners reduces the risk of contracting the virus, but there may be infected people who have had only one sexual partner in their lifetime, but this partner may have had a history of HPV infection. The best method of prevention is sexual abstinence [33].

HPV vaccine is another option of prevention. Certain vaccines can protect individuals against HPV infection and the development of a variety of benign and malignant tumors caused by this virus family. Cervarix, Silgard or Gardasil (Table 2) are three types of safe vaccines that are utilized in the community for prevention [61,62]. These vaccinations are safe to give to both men and women, but do not treat illnesses already present in the host organism [63,64,65,66].

Table 2.

Types of HPV vaccines administered to the population with different age groups [61]

	Cervarix (bivalent HPV vaccine)	Gardasil (4-valent HPV vaccine)	Gardasil 9 (9-valent HPV vaccine)
Type of HPV for which the vaccine provides protection	16, 18	6, 11 (genital warts), 16 and 18	6, 11 (genital warts), 16, 18, 31, 33, 45, 52 and 58
Gender and age range	Females, 9–26 years	Males and females, 9–26 years	Males and females, 9–26 years, 27–45 years (not routine recommended)
Dosing schedule	Three-dose series: 0, 1, 6 months	Three-dose series: 0, 2, 6 months	Three-dose series: 0, 2, 6 months

HPV: Human papillomavirus

Infected patients should counsel their partner to prevent or administer appropriate treatment, because untreated genital warts can progress to malignant lesions [67].

Patients who are infected should counsel their partner on how to avoid or cure genital warts, as untreated genital warts can proceed to malignant tumors [67].

CA detection, on the other hand, can have a significant emotional impact on people affected. It should be emphasized, however, that different treatments can inactivate the virus or cause it to disappear spontaneously, and patients must perform various investigations that test the presence of various STDs and certain immunosuppressive pathologies. Also, infected patients should know that although wart lesions may disappear, they can still transmit the virus to sexual partners [67].

⧉ Histopathological and immunohistochemical examinations

Histopathological (HP) examination of biopsies validates a CA diagnosis. CA is related to acanthosis, parakeratosis, papillomatosis and koilocytosis. Immunohistochemistry can be used to determine the presence of specific HPV strains or antigens. Although no clear causative links between HPV and VC infections have been established, oncogenic activation of the virus is thought to inactivate the p53 tumor suppressor gene and may promoting tumor development [68].

Histologically, VC can vary from pseudoepitheliomatous lesions to SCC. Vacuolation and the presence of keratohyalin granules in the epithelial granular layer structure are both characteristics of genital zarts, but may differ in VC [69,70].

Differentiation between VC and SCC is possible based on the expression of specific oncogenes. Both are positive for immunohistochemical (IHC) labeling with anti-Ki67 or anti-p53 antibodies, but in VC, immunoreactivity is limited to the lower 1/3 of the epithelium, whereas in SCC, immunoreactivity is widespread across the epithelium [71].

Another complication of persistent HPV infection is the development of 2/3 grade cervical intraepithelial neoplasia, which can lead to invasive cervical cancer in 10–20% cases of infected women [72].

Penile cancer can occur in men with HR HPV infection and a history of warty lesions; however, it is 10-fold less prevalent than cervical cancer. According to studies, infected men had a 5.9-fold increased chance of developing penile cancer compared to individuals without a history of warty lesions [73].

⧉ Case presentation

The clinical case we report here is a 29-year-old female, without obstetric history, with a pathological history of epilepsy who was treated with Depakine (three tablets every eight hours). Atypical squamous cells of undetermined significance (ASC-US) result of liquid-based cervical cytology was identified along with the presence of five HR HPV types (16, 33, 45, 59, 68) in extended genotyping. The patient stated that she was sexually abused when she was eight years old and had the first epileptic seizure and is undergoing treatment to date. Additionally, she had cauliflower warts for almost seven years that have not responded to local topical treatment and had an imbalanced life filled with intense sexual activity.

She presented in the polyclinic, to the Service of Obstetrics and Gynecology, Emergency County Hospital, Târgu Jiu, Romania, for the appearance of tumor formations in the vulvar and anorectal regions, with an onset time of roughly 6–7 years and progressive growth in size, discomfort, local pain, and leucorrhea.

Clinical examination of the patient revealed the presence of cauliflower tumors, multiple, with irregular surface, with a single implantation base on each formation, reddish white, with small areas more intensely colored, painful to the touch, in pubic, vulvar, and anorectal region (Figure 1). Following the clinical examination and performing a detailed anamnesis, the patient was hospitalized in the Clinic of Obstetrics and Gynecology to perform specialized investigations and treatment procedures.

Figure 1.

(A–D) Clinical examination of the patient and observation of tumor formations with cauliflower appearance, pale white-pink color, with small areas more intensely colored, bloodless, with pubic, vulvar and anorectal localization, with multiple areas of necrosis, which bleed slightly

At the local gynecological examination, it was found the presence of several tumor formations, of condylomatous appearance, of large dimensions, which include both labia, as well as perineum and perianal, with multiple areas of necrosis, which bled easily. At the valve examination, no lesions of the cervix were detected, macroscopically visible and no blood was lost vaginally.

The local gynecological examination revealed the presence of many tumor formations with a condylomatous form, large dimensions, involving both the labia and the perineum, and perianal regions, with multiple areas of necrosis that bled slightly. No lesions of the cervix and no vaginal bleeding were observed macroscopically during the valve examination. After performing the serological analyzes, it was decided to perform the surgical treatment, with the excision of tumor (Figure 2) and to send the tissue pieces to the Department of Pathology for the HP examination.

Figure 2.

(A) Intraoperative appearance of post-excisional wounds. Areas of surgical suture at the pubic, labial, vulvar and anorectal levels are observed. (B) Excised tumor formations. It is observed the cauli-flower aspect, with a unique development base, with colors ranging from white-pink to more intense areas

The processed paraffin blocks were sent to the Research Center for Microscopic Morphology and Immunology, University of Medicine and Pharmacy of Craiova, Romania.

The blocks were sectioned at 5 μm with the aid of a HM350 rotary microtome equipped with a section transfer to water bath system (STS microM).

The slides were deparaffinized, rehydrated with successive alcohol baths with decreasing concentration of 100%, 96%, 90%, 70% (five minutes each) and then with distilled water (dH₂O) for 15 minutes. The sections have been stained using classical Hematoxylin–Eosin (HE) and Masson’s trichrome (MT).

For IHC studies, antigenic unmasking was performed using a citrate pH 6 or Ethylenediaminetetraacetic acid (EDTA) pH 9 solution; the tissue was inactivated with oxygenated water (H₂O₂) (30 minutes) and non-specific endogenous sites were blocked using skim milk (30 minutes). After tissue preparation processes, the antibodies of interest were prepared in the appropriate concentrations (Table 3), and the slides were placed to 4°C (for 18 hours). The following day, the slides were put at room temperature, washed in phosphate-buffered saline (PBS) solution and the specific secondary antibody was applied for each immunoreaction [mouse/rabbit immunoglobulin G (IgG) antibody, VC002-025, R&D Systems, VisUCyte Horseradish Peroxidase (HRP) Polymer] (one hour). After washing the slides in PBS to remove the secondary antibody, the slides reacted with 3,3’-Diaminobenzidine (DAB) (Dako), and the cell nuclei were stained with Hematoxylin solution. At the end of the working protocol, the slides were dehydrated in progressive alcohol baths of 70%, 90%, 96%, and 100% (five minutes each), clarified in xylene baths (3×15 minutes/bath), and finally, the slides were mounted, so that they could be studied microscopically and stored for a long time without affecting the tissue by contact. The colored slides were scanned at a 20× lens using a Motic EasyScan equipment (Motic China Group Co., Ltd., Xiamen, China) and saved in proprietary format in a database in the Motic Digital Slide Assistant package. They were then exported as *.tiff files at full resolution for further processing.

Table 3.

Immunohistochemical panel of antibodies used by us

Antibody	Manufacturer	Clone	Antigenic exposure	Secondary antibody	Dilution	Labeling
Anti-CK AE1/AE3	Dako	AE1/AE3	Citrate, pH 6	Monoclonal mouse anti-human CK	1:50	Epithelial tissues
Anti-CK5/6	Dako	D5/16B4	EDTA, pH 9	Monoclonal mouse anti-human CK5/6	1:50	Basal cells
Anti-CD34	Dako	QBEnd/10	Citrate, pH 6	Monoclonal mouse anti-human CD34 Class II	1:50	Neoformed blood vessels
Anti-p16	Invitrogen	1D7D2	Citrate, pH 16	Monoclonal mouse anti-p16INK4a	1:50	Human protein CDKN2A – present in HPV infection
Anti-Ki67	Dako	MIB-1	EDTA, pH 9	Monoclonal mouse anti-human Ki67	1:50	Cells in division in the G1, S, G2 and M phase
Anti-p63	Dako	4A4	Citrate, pH 6	Monoclonal mouse anti-human p63 protein	1:50	Nuclear marker
Anti-p53	Dako	DO-7	EDTA, pH 9	Monoclonal mouse anti-human p53 protein	1:50	Nuclear marker
Anti-CD68	Dako	KP1	Citrate, pH 6	Monoclonal mouse anti-human CD68	1:100	Macrophages
Anti-CD3	Dako	Polyclonal	Citrate, pH 6	Polyclonal rabbit anti-human CD3	1:50	T-lymphocytes
Anti-CD20	Dako	L26	Citrate, pH 6	Monoclonal mouse anti-human CD20cy	1:50	B-lymphocytes

CD: Cluster of differentiation; CDKN2A: Cyclin-dependent kinase inhibitor 2A; CK: Cytokeratin; EDTA: Ethylenediaminetetraacetic acid; HPV: Human papillomavirus

On HE slides, these benign lesions have a fibrovascular core covered by squamous epithelium. Next to the vessels in the stroma are also present chronic inflammatory cells. The squamous epithelium changes are acanthosis, parakeratosis, papillomatosis and koilocytosis. Koilocytes are focal and usually present in the upper layers of the epithelium. Koilocytic changes include increased nuclear size and wrinkling, perinuclear halo, and presence of mitotic figures (Figures 3 and 4A). When MT staining was used, it was revealed that the tumor mucosa has a lot of collagen fibers intensely colored in blue (Figure 4B).

Figure 3.

Frontal histological section by CA fragment. It is observed the unique base of implantation of the tumor with arborescent macroscopic appearance and the epithelium with acanthosis and undulated surface. HE staining, ×20. CA: Condyloma acuminatum; HE: Hematoxylin–Eosin

Figure 4.

Histological section by CA fragment: (A) Note the thickened epithelium, with the presence of koilocytes especially in the superficial layers and the parakeratosis; (B) The thickened epithelium is observed, with the presence of koilocytes especially in the superficial layers, of parakeratosis, with an increased density of collagen fibers in the stroma. HE staining: (A) ×100. MT staining: (B) ×100. CA: Condyloma acuminatum; HE: Hematoxylin–Eosin; MT: Masson’s trichrome

IHC studies revealed certain particular aspects of CA, so we observed intense reactivity with anti-cytokeratin (CK) AE1/AE3 antibody throughout the thickness of the epithelium (Figure 5).

Figure 5.

Histological section by CA fragment. The thickened epithelium is observed, with the presence of koilocytes especially in the superficial layers and the intense epithelial immunoreactivity. IHC staining with anti-CK AE1/AE3 antibody, ×100. CA: Condyloma acuminatum; CK: Cytokeratin; IHC: Immunohistochemical

The anti-CK5/6 antibody (Figure 6A) was used to detect the basal layers of the HPV-infected epithelium, the site of virus proliferation and the presence of HPV infection was confirmed by intense epithelial immunolabeling with the anti-p16^INK4a antibody (Figure 6B).

Figure 6.

Histological section by CA fragment: (A) The thickened epithelium is observed, with the presence of koilocytes especially in the superficial layers, and the intense epithelial immunoreactivity at the level of the basal layers; (B) The thickened epithelium is observed, with the presence of koilocytes especially in the superficial layers and intense epithelial immunoreactivity to the anti-p16 antibody. IHC staining with anti-CK5/6 antibody: (A) ×100. IHC staining with anti-p16 antibody: (B) ×100. CA: Condyloma acuminatum, CK: Cytokeratin; IHC: Immunohistochemical

Vessels within the stroma of the tumor were identified using an anti-cluster of differentiation (CD) 34 antibody which reacted with vascular endothelial cells present in the fibrovascular cores (Figure 7A). Anti-Ki67 antibody was used to mark tumor epithelial cells during division, particularly in the basal layers (Figure 7B).

Figure 7.

Histological section by CA fragment: (A) Vessels within the fibrovascular core highlighted by the anti-CD34 antibody that marks vascular endothelial cells; (B) The unique base of implantation of the tumor formation is observed, the epithelium with thickened structure, acanthosis, with undulated surface, with positivity in the basal layer at the immunoreaction with the anti-Ki67 antibody. IHC staining with anti-CD34 antibody: (A) ×100. IHC staining with anti-Ki67 antibody: (B) ×100. CA: Condyloma acuminatum; CD34: Cluster of differentiation 34; IHC: Immunohistochemical

The dividing cells showed basal and parabasal epithelial hyperreactivity, as evidenced by the anti-p63 antibody; This antibody revealed a pan-epithelial reaction (Figure 8A). Additionally, a thicker epithelium with acanthosis, an undulated surface and low positivity in the basal layer is observed when the anti-p53 antibody is used. This antibody highlights few basal epithelial cells that were affected by HPV and showed inactivation of the tumor suppressor gene (Figure 8B).

Figure 8.

Histological section by CA fragment: (A) There is a pan-epithelial hyperreactivity, highlighted by the anti-p63 antibody that marks the cells in division; (B) Rare epithelial cells positive for immunoreaction with anti-p53 antibody is observed. IHC staining with anti-p63 antibody: (A) ×100. IHC staining with anti-p53 antibody: (B) ×200. CA: Condyloma acuminatum; IHC: Immunohistochemical

The immune system’s involvement in lesion evolution is demonstrated by highlighting macrophage cells with an anti-CD68 antibody in the structure of the tumor epithelium and underlying tissue (Figure 9A), T-lymphocytes with an anti-CD3 antibody in the structure of the tumor epithelium and underlying tissue with perivascular conglomeration (Figure 9B), and B-cells with an anti-CD20 antibody in the structure of the tumor epithelium and underlying tissue in a relatively low number (Figure 9C).

Figure 9.

Histological sections through the tumor formation that highlights the involvement of the immune system in the lesion evolution: (A) Macrophages are identified both in the thickness of the tumor epithelium, especially in the basal layers and stroma; (B) T-lymphocytes are identified both in the thickness of the tumor epithelium, especially in the basal layers and stroma, with perivascular distribution; (C) Rare B-lymphocytes are identified, both in the thickness of the tumor epithelium and stroma. IHC staining with anti-CD68 antibody: (A) ×100. IHC staining with anti-CD3 antibody: (B) ×100. IHC staining with anti-CD20 antibody: (C) ×100. CD: Cluster of differentiation; IHC: Immunohistochemical

HP examination revealed condylomatous features, inflammatory process with koilocytic atypia.

⧉ Discussions

Lately, CAs have become more frequent and may be associated with malignancies, due to the spread of HPV throughout the population because of more sexual partners and a lack of condom use [74]. There are over 50 HPV strains, but most often, those involved in the occurrence of CA are HPV 6, 11, 16, and 18 strains. The first two types are generally associated with benign tumors, and the last two with severe forms of dysplasia and even with SCC [74,75].

Strauss et al. [76] confirmed the presence of HPV in the tumor structure using electromicroscopy, in 1949. Oriel & Almeida [77] used the electron microscope to find viral particles in the CA structure [77]. In 1971, Oriel [26] stated that this pathology is sexually transmitted, just like gonorrhea [26]. CA occurs through microlesions produced during intercourse, is exacerbated during pregnancy. It can be spread by a simple touch from one area of the skin to another [78]. In the case presented by us, the patient had been confirmed to have an HPV 6 infection for several years but did not follow any local, excisional or topical treatment, leading to the development of condylomatous areas with significant evolution.

Generally, the diagnosis of CA is made clinically, based on the distinctive symptomatic and macroscopic characteristics of the disease, and HPV genotyping is routinely indicated. The definite diagnosis can only be made with the biopsied or excised tissue samples. HP examination is extremely important to be able to differentiate CA from other tumors even some malignant ones such as SCC, which can coexist. Danish studies have shown that HPV has an increased risk of developing malignancies. In cases with marked atypia, it is necessary to perform special IHC staining to exclude a malignant lesion [79].

The treatment applied can be both local, topical, and surgical excision [36]. In this case, surgical treatment was appropriate because the patient complains of discomfort during defecation and sexual contact, pain caused by compression of the giant tumor and the desire to resume to her sexual life, being a young patient. The surgical treatment was the excision of the tumor formation and suturing the wounds to minimize the possibility of recurrence due to autoinoculation.

From a HP point of view, CA was studied a long time ago, with lesions such as acanthosis hyperkeratosis, parakeratosis, and the presence of modified keratinocytes, called koilocytes [80,81,82]. The performed studies described several types of lesions, partially achieving clinical and HP correlations [83,84,85]. Analysis of numerous biopsies revealed that there are several phases of CA: growth phase, the full-expression phase, and the regressive or persistent phase. These have been described histopathologically as cutaneous skin papillomas by Hamada et al. [86].

It has been observed that only flat warts can regress spontaneously due to immunity, through early infiltration of macrophages and T-lymphocytes into the dermis and subsequently transepithelial. CD4+ cells are the most active, while CD8+ cells are particularly active in lesions that do not regress [87,88,89,90,91]. The case described by us had immunoreactivity for macrophages immunohistochemically labeled with anti-CD68 antibody and T-lymphocytes immunohistochemically labeled with anti-CD3 antibody, particularly in the dermis, with a low number of intraepithelial cells, which may indicate that the tumor became vegetative and was unable to regress spontaneously. In our study, CD20+ B-lymphocytes were present intratumorally in a very low number.

In 1993, Okabayashi et al. [92] managed to describe CA by using rabbits, as proliferative lesions, with dividing and regressive cells, with a lot of intradermal and intraepithelial inflammatory cells.

In 1994, Coleman et al. [16] published their first study with patients who had regressive warts and detected an extensive subepithelial inflammatory infiltrate.

Randomized studies have developed a protocol for HP analysis of CA, aiming to identify the evolutionary phase by the presence of koilocytes, hyperplasia, acanthosis, and hyperkeratosis [93]. The case described by us presented all these lesions and they were emphasized by the classical HE staining.

The example presented here did not spontaneously regress, despite the presence of T-lymphocytes in the dermis, but keratinocytes were detected in varying degrees of cell division, which supported tumor growth and development. These proliferative cells were immunohistochemically labeled with anti-Ki67 antibody. Ki67 antigen is a proliferation-associated nuclear protein [93]. The degree of cell proliferation indicates that the tumor was constantly developing.

The anti-p63 antibody recognizes a protein encoded by the tumor protein p63 (TP63) gene [94] and is a member of the p53 family of transcription factors. It was used to identify cells with dysplastic and neoplastic transformation potential, as demonstrated by numerous studies. Overexpression of the TP63 gene is associated with inactivation of tumor suppressor genes in vulvar cancers [95]. In our case, we observed intense immunoreactivity, which demonstrates the potential for malignant transformation. P63 also contributes to the differential diagnosis of other types of malignancies, as it is strongly positive for SCC and negative for mall cell carcinoma or adenocarcinoma [96].

The TP53 gene encodes the p53 tumor protein, also known as the genome guardian, which prevents genome alterations, being classified as a tumor suppressor gene [97,98,99,100,101,102]. The p53 gene is the most often mutated gene in cancer (>50%) and plays a critical role in the development of neoplasms [103]. In pathologies caused by HPV infections, such as CA, where is encoded a gene, E6, which binds the p53 protein and inactivates it. This phenomenon occurs synchronously with the inactivation of the pRb cell cycle regulator by the HPV-specific E7 protein, leading to cell division and the appearance of warts. HR HPVs, especially 16 and 18, can transform a benign wart into a dysplastic or neoplastic one [104]. In our case of CA, the cellular reactivity for p53 was quite weak, which shows that the tumor had a low degree of evolution to malignancy, but this does not guarantee long-term stability or the absence of subsequent transformation.

Another tumor suppressor is p16, known as p16^INK4a. It is a cyclin-dependent kinase inhibitor 2A (CDKN2A), and also a protein that slows cell division and cell cycle progression from G1 phase to S phase. The deletion of a small portion of the CDKN2A gene leads to p16 inactivation, accelerates the cell cycle, and leads to several cancers [105,106]. P16 is also used as a biomarker in diagnosing high-grade cervical lesions, SCC, cervical cancer, and vulvar cancer. Cancers that overexpress p16 are generally caused by HPV, and those that poorly express p16 are caused by other factors [107]. It is a frequently utilized marker in gynecological pathology, cytoplasmic and nuclear overexpression of p16 is highly related with HPV infection in genital SCC [108]. We observed a significant immunohistochemistry reactivity in our case of CA, indicating HPV infection and the degree of progression to high-grade lesions.

CK AE1/AE3 is a cocktail of CKs 1–8, 10, 14–16, and 19, except for CK17 and CK18, that is used to identify carcinomas, their invasion, and metastases. Additionally, it distinguishes myofibroblasts from smooth muscle cells [109]. In CA, it marked the whole epithelium intensely and did a differential diagnosis with another type of tumor.

CK5/6 is used in IHC studies to label basal or myoepithelial cells within the structure of various organs, being positive in SCC, basal cell carcinoma, transitional cell carcinoma, thymoma, and salivary gland tumors, allowing for the differential diagnosis of benign and malignant lesions [110]. CA’s epithelial origin is also confirmed by the positivity of CK5/6 in the basal layer of epithelial cells.

Also, intratumoral vascularity, proliferating cells and the presence of p16 and epithelial CK immunostaining contributed to increased tumor growth and progression. In our case of CA, we used the anti-CD34 antibody to highlight the endothelial cells of the blood vessels and implicitly the vascular density that supported the tumor evolution. CD34 is a transmembrane phosphoglycoprotein that was first described in hematopoietic stem cells and was later used as a vascular marker in neoangiogenesis [111,112,113,114,115].

⧉ Conclusions

CA is a common pathology that can be prevented by using several preventive measures against the transmission of HPV. Prompt therapy may limit the development of tumors. Clinical features and symptoms might have a significant impact on the patient’s ability to live a normal life. HP and IHC examinations are important for diagnostic and may reveal various tumor features, such as the presence of increased vascularization, degree of cell proliferation, epithelial affiliation, inactivation of tumor suppressor genes, presence of HPV infection and involvement of the cell-mediated immune system, which can accelerate tumor evolution and development from benignity to malignancy.

Conflict of interest

The authors declare that they have no conflict of interests.