Presence of circulating tumor cells in a patient with multiple invasive basal cell carcinoma - a case report

Introduction

Basal cell carcinoma (BCC) belongs to the group of non-melanoma skin cancer and it is the most common cutaneous malignancy at all [1]. Examples of other non-melanoma tumors include squamous cell carcinoma, Merkel cell carcinoma or sebaceous carcinoma; however, BCC accounts for 80–85% of this category [1,2]. The worldwide incidence of skin malignancies (melanoma along with non-melanoma skin cancer) is epidemically increasing [2,3].

Even though BCC is known to be a slowly growing and very rarely metastatic skin cancer (with an estimated incidence of 0.003–0.550%), the tumor may be destructive to surrounding tissues, and even to a cartilage or a bone [4]. BCC can be easily misdiagnosed for va­rious similar benign skin conditions, and most of them might be underestimated.

Despite the increasing incidence of skin cancer, which may be due to better diagnostic methods, research of li­terature on skin cancer confirmed the rising trends of its prevalence [5]. Circulating tumor cells (CTCs) shed as entire cells and cell clusters from the primary and/or secondary tumors are able to ­migrate into the bloodstream while often having the potential of creating distant metastatic lesions [6].

CTCs are a promising biomarker of malignancy and tumor dissemination. Studies declared potential use of CTCs in the detection, disease staging and mana­gement of metastatic melanoma [7]. A therapy response could be mo­nitored in melanoma patients recei­ving interferon therapy or other immunotherapies, such as immune checkpoint inhibition [8]. As metastatic BCC is exceedingly uncommon, very poor understating of its incidence, risk factors, dissemination and treatment options are known [9]. In accordance with the advanced clinical stage of the reported patient, CTCs isolation has been tested in this case.

Case report

A 67-year-old Caucasian woman was admitted to a Czech district hospital in December 2017 for anemia-induced fatigue and collapse. Anemia, with admission hemoglobin level 4.7 g/dL was caused by chronic bleeding from extensive skin tumors.

In the year 2012, she had already undergone excision of small BCC on the trunk and scalp regions. After the appearance of new lesions, she was afraid of undergoing another surgical procedure. She was bandaging the growing multiple ulcerated skin lesions and hi­ding them from her relatives.

The patient was thoroughly examined by medical imaging; X-ray of the chest, ultrasound of the abdomen, CT scans of the head and trunk, gastroscopy and colonoscopy. None of the examinations showed any other cause of bleeding or metastatic involvement. After all of the examinations and treatment with blood transfusions, she was recommended to undergo the surgical treatment of skin tumors at the department of plastic and reconstructive surgery.

This patient was presented to our department at the beginning of January 2018 with extensive bleeding skin neo­plasm with a past medical history of atrial fibrillation, diabetes mellitus, hypertension, and hyperlipidemia; her surgical history included total abdominal hysterectomy with bilateral salpingo‐oophorectomy. Clinical examination showed multiple (at least 10) ulcerated skin lesions. Two of them, which were the biggest ones, both on the back, were actively bleeding. The diameter of both bleeding masses was 8–9 cm and around 2 cm above the skin surface (Fig. 1). There were other ulcerated lesions on the neck and parietal region of the head. Other parts of the body, mainly the back, were covered by lesions with the appearance typically described for superficial basal cell carcinoma; mostly scaly, erythematous patches or thin papules [10].

A multiphase surgery was planned with the primary goal of removing bleed­­­ing lesions. The patient underwent the first surgery under general anesthesia, in which wide excisions of the two biggest masses on the back and local flaps for coverage of the defects were performed. We excised three other smaller lesions. Three days later, we continued in surgical removal of other suspicious lesions under local anesthesia. Three months later, after the patient’s reco­very, we proceeded into the next phase of the removal of larger lesions under general anesthesia. The BCC of the scalp expanded virtually 1% of the total body surface area. A wide excision of the ulcerated scalp mass was performed, and the defect was covered by a split-thickness skin graft. During this operation, smaller presumed skin tumors were removed by excision. The patient healed very well without any complications.

All 14 excised lesions were sent for histopathological examination, which showed a combination of invasive, no­dular and superficial BCC with clear margins except for one area of the left temporal region. Despite our efforts of surgical elimination of multiple BCCs, we observed rapid growth of previous lesions and the occurrence of new lesions predominantly localized on the back and the head. In agreement with the oncologist and the dermatologist, we came to the conclusion that the patient could no longer be offered further surgical therapy or local dermatological therapy.

The patient was referred to oncologists for further treatment and monitoring. Radiotherapy had not been indicated due to the extensive affected skin areas. Genetic examination verified Gorlin syndrome (nevoid basal cell carcinoma syndrome). Targeted therapy with a hedgehog pathway inhibitor had been initiated. From the beginning, the treatment was without any complications and a reduction of some lesions was observed. Later on, complete regression of several BCCs was confirmed. The patient had been treated with Erivedge (Erivedge®, Vismodegib, Genentech, USA) 150 mg once a day for almost a year with mild side effects only (occasional diarrhea and hair thinning). This therapy will continue until drug toxicity.

Circulating tumor cell investigation

As previously mentioned, BCC is known for its individual invasive local growth. The patient described in the study presented multiple and rapidly growing skin lesions with an intention of syste­matic spreading. Isolation of CTC was indicated to prove the possible systemic disease character following a thorough examination which excluded other malignancies that could affect the results.

After the patient signed the informed consent, she was examined for CTCs throughout the entire period of her treatment. The patient underwent regular blood sampling, approximately 8 mL of venous blood was drawn from the cubital veins and placed into S-Monovette tubes (Sarstedt AG & Co., Numbrecht, Germany) containing 1.6 mg EDTA (ethy­lenediaminetetraacetic acid) /mL blood as an anticoagulant. The samples were processed at room temperature using an isolation procedure completed within 24 hours after blood draw.

CTCs enrichment and culture: A size-based separation method for viable CTC-enrichment from peripheral blood was used (MetaCell®, MetaCell s.r.o., Ostrava, Czech Republic) [11]. The size-based enrichment process is based on the filtration of peripheral blood through a porous polycarbonate membrane with pores of 8 μm in diameter. The stan­dard 8 mL of peripheral blood were transferred to the filtration tube. The perip­heral blood flow is supported by the capillary action of the absorbent touching the membrane filter. The membrane filter, which is kept in a plastic ring, was transferred directly into a 6-well culture plate and 4 mL RPMI (Roswell Park Memorial Institute) medium con­taining 10% FBS (fetal bovine serum) was added to the membrane top and CTCs were cultured on the membrane in vitro under standard cell culture conditions (37°C, 5% CO₂ atmosphere) for a minimal period of 7 days on the membrane. Vital fluorescence stains (NucBlue® Live ReadyProbes® Reagent, CellTracker™ Green CMFDA, MitoTracker Red CMXRos, all by ThermoFisher Scientific, USA) were applied on the cultured cells to visua­lize cell nucleolus, cytoplasm, and mitochondria. The membrane with the captured cells was transferred to a microscopic slide and examined using fluo­rescence microscopy at magnification 20× to get an overall view of cells on the membrane and further at magnification 40× and 60× for detailed cytomorphological analysis. Isolated cells and/or clusters of cells of interest were selected, digitized and examined by an expe­rienced researcher.

Blood sampling was initiated in the morning before the first of the surgeries in January 2018 (Fig. 2). The collection of blood specimens was obtained from cubital veins under aseptic conditions. The first samples showed the presence of CTCs in an uncountable amount, more than a thousand cells on the filtration membrane were detected. The cytomorphological analysis showed cells with epithelial character of regular shape, smooth nucleus structure and regular nuclear contour (Fig. 3)

An additional blood test was performed before the second stage of skin cancer removal, about 3 months after the primary surgery in April 2018. In this phase of lesions elimination, a small section of tumorous tissue was sampled and sent along with blood for comparative cellular examination. The results surprisingly presented a significant reduction in the number of CTCs to a level that was the lowest throughout the whole measurement. There were only 20 cells on the membrane. The cells from the primary tumor cultivated during the same period, stained as examined CTCs from the peripheral blood, showed the same morphological characteristics, thus explaining their origin in the circulation. During this period, when the largest ulcerated bleeding tumors were surgically removed, our patient was in a stable stage regarding the constant number of tumor lesions, which once again corresponds to the lowest rate of the mea­sured CTCs.

Two weeks later, in May 2018, the third blood examination was done when the wounds were nearly healed. The number of CTCs steeply rose to about 800. We observed that new BCC lesions began to appear, and previous lesions had grown in size. Shortly afterward, Erivedge treatment in the oncology department had been initiated. Other blood samplings were completed 5 months after the previous collection in September 2018. The wounds after the excisions were completely healed at that time and improvement such as decreased tumor growth due to oncologic treatment was already observed. The presence of CTCs decreased slightly, approximately 500 cells were detected. The last blood sample was taken 9 months after therapy ini­tiation, in March 2019, when the patient had no more complaints and her quality of life improved significantly. The number of CTCs reached almost the lowest previous value, 50 cells on the membrane were identified.

Discussion

In the past two decades, big efforts and expectations were put into the disco­very of new non-invasive tools for diagnosis of cancer. Further improvement of these markers could help in more precise diagnosis, specific prognosis, personalized therapy and early recurrence alert. Examination of CTCs, known also as liquid biopsy, is easily collected from the patient and allows to examine molecular architecture and behavior of tumor cells in real time. Thus, it helps to understand disease pathophysiology and gives a chance to promptly react to treatment effectiveness [12]. CTCs are cells which are shed from the primary tumor, enter the bloodstream of the patient and have the potential to develop distant metastasis [13]. Positive isolation and detection of CTCs has been va­lidated as a prognostic factor in metastatic breast cancer and several other solid tumors such as prostate, colorectal and lung cancer [14].

This case report shows the presence of CTCs in a patient with BCC, a malignant cancer known for its local invasive growth instead of systemic spread. No study evaluating a BCC tumor and the presence of CTCs in peripheral blood has been found so far.

Before the first therapeutic surgery, the number of CTCs was extremely high. This is very interesting due to the fact, that the general presence of CTCs in patients with malignancy is rather low and these high numbers could be matched with blood samples from heavily disseminated metastatic diseases.

Our case shows that tumor removal itself influenced the reduction of the CTCs number. The question is whether the removal of all skin malignancies would lead to complete elimination of CTCs. In our patient, a significant re-growth of CTCs was recorded even after the removal of most of the BCCs, up to the treatment with a hedgehog pathway inhibitor causing CTCs rate decline. These findings suggest that we should keep in mind the possibility of early progress of BCC into a systemic disease. Although BCC is commonly referred as a local di­sease, in advanced cases systematic therapy with hedgehog inhibitors is used with a positive outcome, which is also in correlation with the presented case. With the appearance of new BCC lesions, an increase of CTCs in peripheral blood was observed. The growth of these new lesions has ceased only through permanent reduction of CTC cells in the blood.

Conclusion

We report a case of successful isolation of CTCs in multiple invasive BCC with dynamic change of cell quantity reflecting the surgical and oncological treatment. While surgical excisions were followed by an immediate, although temporary drop in cell count, a final persistent decrease was achieved with hedgehog pathway inhibitor treatment. This continual decline of CTCs was eventually associated with termination of developing new lesions and regressing the existing ones. The detection of CTCs in this case points to a possible systematic behavior. Surely, there is a requirement for further studies and approval of the presence of CTCs in other patients who are treated for BCC.

Acknowledgements: This work was funded by the Charles University Grant Agency, Progres Q40/04.

Ethical approval: All procedures performed in the case study involving human participant were in accordance with the ethical standards of the institutional research committee (Ethics Committee, University Hospital Hradec Kralové, Sokolská 581, 500 05 Hradec Kralové, Czech Republic, reference number 201805 S09P) and with the Helsinki declaration from 1975 (revised in 2000) and its later amendments or comparable ethical standards.

Roles of authors: All authors contributed equally to preparing the manuscript.

Conflict of interest: The authors state that there are no conflicts of interest regarding the publication of this article.

Katerina Kiss, MD

Průběžná 1826/63, 100 00 Prague 10

Czech Republic

e-mail: kiss.katerrina@gmail.com

Submitted: 04. 10. 2020

Accepted: 27. 02. 2021