Current Challenges in the Association between Canine Leishmaniasis and Malignant Disorders

Abstract

Neoplasms in dogs are estimated to account for up to 83% of all canine malignancies, while canine leishmaniasis is endemic and highly incident in areas like Brazil, east Africa, and India. The worldwide incidence of both diseases may indicate the presence of these two pathologies in the same tissue sample as eventual cases. However, a relationship between leishmaniasis and malignant disorders has been postulated in human and veterinary medicine. Depending on several direct or indirect factors, leishmaniasis can affect the clinical manifestation, diagnosis, therapeutic protocols, and the outcome of various malignant disorders. In addition, the similarity of the clinical presentation of cutaneous or visceral leishmaniasis with the clinical signs observed in many tumors may lead to misdiagnosis. Clinical cases of co-localization of Leishmania parasites and tumor cells in dogs were more associated with transmissible venereal tumors than histiocytic tumors, non-histiocytic lymphoma, or squamous cell carcinoma. This chapter reviews the circumstances involving canine leishmaniasis and malignant disorders, and the current challenge that the association between them represents to misdiagnosis or tumor-promoting potential of leishmaniasis, providing an overview of dog cases.

1. Introduction

Neoplasms in dogs are estimated to account for up to 83% of all canine malignancies [1]. Its etiology is largely unknown and believed to be multifactorial [2], genetic [3, 4], auto-immune [5], hemoparasitic [6, 7], and viral diseases [8]. The exact mechanism by which chronic inflammatory processes induce oncogenesis is still unclear, but persistent immunosuppression [9], epigenetic events [10], and relevant association with immunosenescence [11] have all been associated with neoplasm developments.

Leishmania infantum-infected macrophages parasitize tissues commonly causing granulomatous inflammatory reactions [12]. Infected dogs with poor body condition, or cachexia, mainly display skin signs including skin peeling, cutaneous lesions (nodular, ulcerative, and pustular), and exfoliative dermatitis. A wide variety of clinical signs and a number of unusual presentations make diagnosis a challenge to veterinarians [13, 14, 15, 16].

L. infantum invades most of the dogs tissues and organs [16, 17] and it has also been sporadically reported in association with different types of tumors [7, 18, 19], including cutaneous large-cell lymphoma [20], cutaneous canine transmissible venereal tumor [21], splenic [20], hemangiosarcoma [22], soft tissue sarcoma, cutaneous small-cell lymphoma, adrenocortical adenoma [7], and perianal adenoma [23].

In transmissible venereal tumor (TVT), sarcoma, small-cell lymphoma, and adrenocortical adenoma, amastigotes were observed in the macrophage’s cytoplasmic vacuoles and inside the neoplastic cells, probably in consequence of an atypical dissemination of Leishmania parasites [23]. These findings demonstrate the ability of Leishmania to parasitize neoplastic cells other than leucocytes [7, 19].

A relationship between leishmaniasis and malignant disorders has been postulated in human and veterinary medicine [22, 24]. Among the proposed theories, the chronic inflammation induced by Leishmania persistence, the interference with the local systemic immune system, and a direct implication of the parasite in the pathogenesis of cancer are considered most important. Etiopathogenic theories are based on the pathological observation that chronic inflammation can lead to development of dysplasia, abnormalities in cell division and activation of the p53 protein (an important tumor suppressor protein, whose loss of function leads the cell to genomic instabilities, i.e., the tumor sets in) [25]. However, definitive evidence of this association has not been proven to date. Depending on several direct or indirect factors, leishmaniasis can affect the clinical manifestation, diagnosis, therapeutic protocols, course, and outcome of various malignant disorders. The association between leishmaniasis and malignancy can be identified in four distinct situations: leishmaniasis mimicking malignancy, leishmaniasis associated with malignancy, malignancy developing in patients with leishmaniasis blemish, and leishmaniasis developing in patients with malignancy [24]. This chapter reviews these situations involving canine leishmaniasis and malignant disorders, and the current challenge that the association between them represents to misdiagnosis or tumor-promoting potential of leishmaniasis, providing an overview of dog case reports, and when possible, draws a comparison with case reports in humans.

2. Histopathology, biochemical, and hematological parameters of canine leishmaniasis

The liver, spleen, and lymph nodes are the organs most affected by the disease [26], as well as the kidney and skin. In humans with visceral leishmaniasis, liver function and morphology are modified by the presence of an inflammatory process in the portal and intralobular region, locally concentrated or diffusely localized. In dogs experimentally infected by intravenous and intraperitoneal injection of amastigotes of L. infantum, Gonzalez et al. [27] verified that, in both forms of inoculation, the animals displayed alterations with multiple granulomas, with the visualization of the parasite, without predilection location. In addition to the Kupffer hypertrophy and hyperplasia are often seen in dogs naturally infected. Diffuse intralobular liver fibrosis was reported by Melo et al. [28], demonstrating that there are differences between infected and non-infected, with collagen deposition in symptomatic animals when compared to asymptomatic ones. When evaluated for the presence and size of granulomas, asymptomatic and symptomatic animals did not show differences between groups [29]. There is a granulomatous infection that only infiltrates Kupffer cells and hepatocytes. In chronic infections displaying different levels of severity, the granuloma was limited to the sinusoids and expanded to the portal region and the capsule, forming diffuse areas in resistant dogs. Hepatic granuloma in dogs affected with leishmaniasis is not organized [30].

Parasitism caused by L. infantum in the spleen can be verified, especially in the capsular and subcapsular regions and in the marginal zone, with mild intensity in asymptomatic animals, and intense parasitism in symptomatic ones [26, 31]. Studies demonstrated a reduction in regulatory T cells in the splenic tissue, indicating that they are capable of producing a greater amount of IL-10 when compared to non-infected dogs [32]. Santana et al. [29] found that susceptible dogs, that had a Montenegro intradermal reaction negative and splenic culture positive, showed smaller and fewer follicles, as well as changes in the splenic follicular organization, while severe form of leishmaniasis is associated with disorganization of white pulp [33].

In the kidney, the accumulation of immune complexes in the glomeruli can initiate glomerulonephritis and interstitial nephritis, which can lead to chronic renal failure in dogs, [34]. Santos et al. [35] evaluated histological alterations of dogs naturally infected with Leishmania infantum and observed amastigote form of the parasite in the kidneys and bladder of infected dogs, demonstrating that the clinical signs resulting from the urinary system alterations should be taken into account when diagnosing this disease. In fragments of the urinary bladder main alterations observed through the histopathological analysis were inflammatory reactions in the adventitial layer, characterized by moderate mononuclear infiltrate and focal perivascular infiltrate. The kidney showed glomerular degeneration and atrophy, granulomatous inflammation, apoptosis, and Bowman capsule lesions.

In general, skin lesions in dogs are focal ulcers, with raised edges, like human lesions. Other dermatological signs are alopecia, papular, and nodular dermatitis [36]. Boechat et al. [37] verified the presence of L. (L.) infantum amastigotes in the endometrium, ovaries, vaginal mucosa, skin, and mucosa of the vulva and mammary glands, and it was even possible to observe a greater parasite load and intense reaction inflammation in the vulva and breasts of naturally infected dogs. The number of foci and cellularity of the inflammatory infiltrates in symptomatic dogs is higher than in other groups conform seen by Verçosa et al. [38], as well as average area, perimeter, and extreme diameters of the inflammatory infiltrates were higher in symptomatic animals.

Canine leishmaniasis is also associated with alteration in hematological and biochemical parameters. Erythrogram of dogs seropositive for leishmaniasis shows moderate anemia, usually presented in the normocytic and normochromic form with non-regenerative character [39, 40]. This characteristic can be a consequent of the bone marrow invasion by the parasite, which induces inflammation and contributes to the reduction of erythrocyte production [41]. A thrombocytopenia is common and occurs due to vasculitis caused by immune complexes circulating blood, thrombocytopoiesis disorders, and platelet destruction [40].

Leukocyte response changes according to the stage of the disease [42]. Lymphopenia occurs due to the sequestration of cells in the spleen and lymph nodes, as well as by the destruction caused by Leishmania [43]. Other common changes such as eosinophilia and leukopenia are associated with clinical disease [44]. Lymphocytosis was observed in asymptomatic dogs [16, 45], biochemical parameters levels in serum may be higher than in dogs seronegative [43], and, in some cases, severe proteinuria may determine changes in normal serum protein values [46].

An unusual case of persistent pancytopenia caused by leishmaniasis in a human patient receiving immunomodulatory therapy for myeloma was recently described [47] and draws attention to possible similar cases in dogs. Pancytopenia in multiple myeloma is commonly caused by plasma cell infiltration and cytotoxic medications, however, fungal, bacterial, or viral infection, and treatment-associated myelodysplasia also warrant consideration. In regions where leishmaniasis occurs naturally, it should be also investigated, and in this case, the diagnosis of Leishmania infection and a proper therapeutic can reverse pancytopenia.

An usual alteration associated with leishmaniasis is hyperproteinemia, caused by the activation of B lymphocytes and high production of antibodies [40]. In fact, there is a decrease in the serum albumin level, in contrast to an expressive increase in the globulin levels, causing a significant increase in the levels of total plasmatic proteins during canine leishmaniasis [43, 48]. Liver function may also be normal or with changes suggestive of liver diseases [49]. It can be detected by increased activity of liver enzymes such as alkaline phosphatase (AP) and alanine aminotransferase (ALT) [49, 50, 51].

Alterations in biochemical parameters can also be associated with leishmaniasis treatment. A case report of a tumor lysis-like syndrome during therapy of visceral leishmaniasis consisting of hyperkalemia, hyperphosphatemia, hyperuricemia, and acute renal insufficiency, soon after the initiation of liposomal amphotericin B chemotherapy for severe visceral leishmaniasis, was observed in a human patient. Allopurinol therapy and intravenous fluid administration lead to a full recovery of the metabolic parameters. Awareness of this condition by veterinarians can lead to early recognition and preventive management of patients undergoing leishmaniasis treatment [52].

3. Tumor and Leishmania infection association

The worldwide incidence of cancer and the endemicity of leishmaniasis in areas like Brazil, east Africa, and India may indicate the presence of these two pathologies in the same tissue sample as eventual cases. Both diseases display an immunological bias in the pathology, course, and outcome of the disease progression associated with an impaired host Th1 response. Therefore, the Th2 cytokine microenvironment occurring in compartmentalized organs in progressive leishmaniasis [53] may potentially promote tumor cell proliferation as well as the other way around. In addition, the similarity of the clinical presentation of cutaneous, mucosal, or visceral leishmaniasis with lesions observed in many malignant disorders may lead to misdiagnosis [54]. Clinical cases of co-localization of Leishmania parasites and tumor cells in dogs were mainly associated with TVT or less frequently with histiocytic tumor, non-histiocytic lymphoma, or squamous cell carcinoma (Figure 1). First, we will discuss about the less frequent association of tumors with leishmaniasis, as well as misdiagnosis related to both diseases, and then the association of tumors with TVT will be reviewed.

Figure 1.

Organs affected by canine leishmaniasis, and most frequent neoplasms associated with leishmaniasis in dogs.

The clinical identification of leishmaniasis is frequently related to skin lesions. However, most of the evidence about the association between cutaneous leishmaniasis and skin cancer is still based on case reports and other clinical observations rather than strong epidemiological observational studies [55]. An unlikely association of squamous cell carcinoma with an active cutaneous leishmaniasis was reported in an immunocompetent human patient [25], and a report of a case of basal cell carcinoma (Clark level IV) that had been developed on characteristic active lesion of mucocutaneous leishmaniasis, suggesting that carcinogenesis, in this case, was triggered by Leishmania parasites and induced by sun’s ultraviolet (UV) radiation exposure [56]. Although causality is still to be proved, a biopsy may be essential to rule out associated skin malignancy in reluctant cutaneous leishmaniasis [25]. A report of a solitary cutaneous leiomyoma mimicking leishmaniasis demonstrates that neoplasms mimicking cutaneous leishmaniasis are an alert for an appropriate differential diagnosis [57].

Differential diagnosis of leishmaniasis and malignancy disorders due to similarities in clinical presentation, as well as its association, is perhaps reported for the three clinical forms of leishmaniasis: visceral, cutaneous, and mucosal. A chronic skin lesion histopathologically compatible with squamous cell carcinoma, and hence programmed for Mohs surgery, showed to be cutaneous leishmaniasis after review of the clinical and epidemiological history, and laboratory diagnosis. Treatment with miltefosine led to a complete resolution of the lesion [58]. Zambarbieri et al. [19] reported a case of Leishmania spp. infection in a cutaneous histiocytoma in a nodular lesion on the metacarpus of an old dog. In cytopathology examination, in some neoplastic cells, with moderate anisocytosis and anisokaryosis, Leishmania spp. amastigotes were observed in the cytoplasm.

A report of visceral leishmaniasis in a human patient with a lung tumor highlights leishmaniasis as a possibility to be considered in the diagnosis of causes of fever in patients undergoing chemotherapy in an endemic area [59]. In humans, visceral leishmaniasis was also reported in association with Hodgkin’s lymphoma [60], melanoma [61], and lymphoma, the last with cases of Leishmania infection or clinical manifestation after treatment for cancer [62]. The visceral leishmaniasis in association with multiple myeloma highlights the importance of histopathological correlation of positron emission tomography (PET) findings [63]. In this case, the liver, spleen, and bone marrow are the sites of infiltration most frequent in visceral leishmaniasis and should be observed as a differential diagnosis in PET scans.

A mucosal lesion with a tumor-like appearance by indirect laryngoscopy, but negative in indirect immunofluorescence assay test and bone marrow aspirate examination for Leishmania, revealed in biopsy a granulomatous inflammatory infiltrate and abundant Leishmania amastigotes, in addition to Leishmania parasites growth in culture, in a human patient. The lesions disappeared after treatment with meglumine antimoniate (850 mg/day) for 28 days [64]. Atypical cases of mucocutaneous leishmaniasis in human mimicking rectal cancer reported two different presentations: an erythematous nontender plaque measuring 5 cm by 5 cm was observed over the right perianal area of a 32-year-old male HIV patient; and two masses of different sizes and characteristics were observed around the anus and a fungating circumferential mass of 8 cm was seen above the proximal anal verge in a 40-year-old male. Atypical mucocutaneous leishmaniasis should be considered as a possible diagnosis in patients with chronic skin lesions resembling hemorrhoids and colorectal masses, especially in endemic areas for leishmaniasis [65]. A 50-year-old man mimicking multiple myeloma, presenting pancytopenia, hypergammaglobulinemia with concurrent hypoalbuminemia, showed normal cellularity, mild hemophagocytosis, increased plasma cells, and Mott cells in bone marrow aspirate. However, a thorough inspection of the bone marrow smears revealed the presence of Leishmania parasites. Mott cells are plasma cells with multiple cytoplasmic inclusions of immunoglobulin (Russell bodies) and it was associated with multiple myeloma cases. However, they have also been found in patients with infections such as Leishmania infection [66].

Other atypical report is the co-occurrence of mucosal leishmaniasis caused by Leishmania infantum and a retrospectively diagnosed concomitantly with a marginal zone lymphoma (MZL) in a 62-year-old man tongue. The patient was successfully treated first for MZL, but after six months, the tongue swelling returned with the identification of Leishman bodies (Giemsa stain) within the macrophages in the biopsy sample. Liposomal amphotericin B induced clinical improvement [67]. A human patient with metastatic non-small-cell lung carcinoma, after chemotherapy and a high dose of corticosteroids, showed a painful and swollen tongue. Biopsy showed an inflammation with histiocytes and Leishmania amastigotes indicating that the lowered immune status was important in developing mucosal leishmaniasis [68].

A gingival tumor-like lesion measuring 3 cm in the longest axis was reported to be an atypical presentation of visceral leishmaniasis in a six-year-old male mongrel dog. Upon histopathological analysis, a granulomatous inflammatory infiltrate, composed of plenty of plasma cells, lymphocytes, and histiocytes filled with amastigotes, was observed in the biopsy lesion. The diagnosis was confirmed by serological and immunohistochemistry assays. This case report reinforces that clinical and pathologist veterinarians should include leishmaniasis in the differential diagnosis of tumors and other chronic diseases of oral mucosa, especially in areas where the disease is endemic [69].

The relationship between parasites and immunotherapy is controversial, especially in leishmaniasis. It is well known that the increasing use of immunosuppressant medication by antitumor therapies makes patients susceptible to a new infection or turn symptomatic those asymptomatic Leishmania infected patients. On the other hand, in an experimental breast cancer model in vivo, Leishmania spp. infection demonstrated an immunostimulatory effect in cancer treatment, triggering a strong cellular immune response, determined by an increase in the production of IFN-g and IL-2 and polarized toward Th1, as necessary in the defense against tumors [70].

4. TVT and Leishmania infection association

Canine TVT (CTVT) is a unique neoplastic entity, contagious and sexually transmitted especially those of street life and of reproductive age, and regarded as the oldest known mammalian somatic cell neoplasm in constant transmission. CTVT occurs worldwide with higher incidence in tropical areas, and has been mostly reported in dogs (Canis familiaris) and foxes (Vulpes sp.) [71, 72]. Due to the species' communication and licking habits, it may occur in different parts of the body as around the eyes, mouth, and nasal cavity, when it is called extragenital TVT [73]. When tumors and/or their metastases occur in extragenital regions, other clinical signs may be present depending on the location of the affected organ [74, 75, 76], such as respiratory disorders, dyspnea, abdominal pain, and dysphagia [73, 77, 78].

Clinical signs often consist of genital lesions which, when present, may manifest as a single or multiple mass, friable on the foreskin or vulva [76, 77, 79], serosanguineous secretion, odor foul, and areas with or without necrosis [73]. In a case reported por Duzanski et al. [79] dog’s tumor initially adhered to the adjacent tissues, and developed to a friable hyperemic mass of the cauliflower aspect. Metastasis may occur with lymphatic or visceral spread usually associated with underlying immunological impairment. In addition, TVT extragenital lesions, such as cutaneous, are common and have been reported even without primary genital lesions [71].

Atypical presentations of TVT are reported in association with Leishmania infection. Albanese et al. [21] reported an atypical primary cutaneous extragenital canine TVT with Leishmania-laden neoplastic cells in three subcutaneous round alopecic nodules, approximately 6–8 cm in diameter, located on the anterior and caudal dorsal region and in the ventral area of the neck. Intracytoplasmic L. infantum amastigotes were observed in the infiltrating macrophages. Intracellular amastigotes, 1–3 per cell, were present in vacuolated tumor cells. The patient received fluid therapy, erythropoietin 100 UI/kg, and intravenous injection of 0.075 mg/kg vincristine presenting regression of the nodules that decreased in size after one week, however, euthanasia was necessary due to a progressive worsening of the general condition of the dog.

A TVT was curiously reported associated with cutaneous metastasis in a female dog with leishmaniasis reactive left prescapular lymph node and subcutaneous nodular mass, not adhered to the musculature. Other visible signs were onychogryphosis, opaque, shineless, and brittle fur, with pruritic and lichenified exfoliative dermatitis in the left scapular region, ear tips and snout, and the presence of a few quantity of brownish-colored vulvar discharge with a putrid odor were the main clinical alterations. Cells with characteristics of TVT located in the genital mucosa, observed by cytology, demonstrated the presence of amastigote forms of the protozoan Leishmania sp. in the interior of the macrophages. TVT cells developed atypical metastasis in the cutaneous tissue in the region of the thirteenth rib. Due to the non-responsiveness of treatment and worsening of the clinical signs, the animal was euthanized [80].

Another atypical presentation of disseminated TVT associated with leishmaniasis occurred in an 8-year-old female dog that was pluriparous. There were no external genital lesions, but TVT cells were present on the serous and endometrial surface in the histopathological examination of the uterine horns, in addition to cystic endometrial hyperplasia. The history of breeding with a stray dog indicates TVT sexual transmission, and then dissemination to the omentum, peritoneum, spleen, and liver, probably favored by immunosuppression [81].

Kegler et al. [82] made the first report describing infection of tumor cells by L. infantum in a genital TVT from an asymptomatic 2-year-old female boxer dog, with a vaginal serosanguineous discharge. Cytological and histological examinations were performed on a friable mass occupying the upper caudal part of the vagina revealing a monomorphic population of neoplastic round cells confirming to be canine TVT, with a presence of L. infantum amastigotes in tumor tissue. The authors highlight the transplantation of Leishmania-laden neoplastic as an alternative route of venereal transmission of leishmaniasis among dogs.

Leishmania sp. amastigotes were also detected in canine TVT of a 10-year-old, mixed breed, intact female dog, with no other clinical abnormalities otherwise. Tumor tissue imprint smears, cytological, and immunohistochemistry examination revealed Leishmania sp. amastigotes within infiltrating macrophages, confirmed by polymerase chain reaction (PCR). Histopathology of the lesion showed extensive infiltration of lymphocytes and plasma cells within the subepithelial vaginal stroma, with numerous neutrophils and macrophages with intracytoplasmic amastigote, and rare reminiscent canine TVT neoplastic cells within reactive area of fibrosis with newly formed collagen [71]. The authors discuss about the possibility of Leishmania infection may have started on or from the canine venereal tumor tissue, the latter option suggesting evidence of an alternative vector-independent route of transmission for canine visceral leishmaniasis in places where both diseases coexist.

Clinicopathological findings of canine TVT in leishmaniotic dogs can aid in the understanding of the biological behavior of TVT associated with leishmaniasis for future cases. In summary, TVT can harbor a large number of Leishmania parasites, and canine leishmaniasis may lower the immune defense against malignancy [83].

5. Conclusions

The cases of tumors that mimic leishmaniasis lesions indicate the need for an adequate cytological and histopathological diagnosis. Likewise, cases of clinical presentation of leishmaniasis that resemble tumors require special attention and may be related to atypical Leishmania species in a given region, as well as host-related conditions, such as immunosuppression. The association between leishmaniasis and tumors may mean that regular diagnostic methods are not sufficiently adequate. Therefore, the use of robust diagnostic tools, with greater precision and accuracy, such as molecular tests to identify the parasite DNA through PCR, can be used to avoid a misdiagnosis. In the same way, the association between leishmaniasis and tumors is a challenge as they can arise due to cases of immunosuppression induced or not by treatment, leading to hematological and biochemical changes that can compromise the effectiveness of the treatment. Atypical conditions related to the clinical presentation of tumors or leishmaniasis in dogs require special attention, especially in endemic areas, where the probability of association of both diseases becomes greater and should always be taken into consideration in the management and conduct of the patient in human and veterinary medicine.

Acknowledgments

This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (Finance Code 001), by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (26/211.680/2021) and Fundação de Amparo à Pesquisa e Desenvolvimento Científico e Tecnológico do Maranhão (APP-12233/22). HSR (CNPq 150336/2023-3) and FA-S (CAPES 88887.363006/2019-00) are postdoctoral research fellows. KC (315225/2021-1) and ALA-S (313348/2021-9) are research productivity fellows by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq.