By Rosa Ferrer, Andrea Mansilla and Maria Costa

The role of human chorionic gonadotropin in pregnancy has been extensively studied for many years. However, the importance of this hormone goes beyond pregnancy. It can also be used as a biomarker, mainly in trophoblastic tumours, but as will be shown later in this article, its importance in non-trophoblastic tumours is on the rise.

hCG: biochemical structure, subtypes and biological activity

Human chorionic gonadotropin (hCG) is a glycoprotein hormone physiologically produced by trophoblastic cells in the placenta during pregnancy. It is a crucial tool for monitoring pregnancy, as we all know. Besides this, it has been demonstrated that both trophoblastic and non-trophoblastic tumours generate a quantity of hCG molecules.

Structurally, it is a heterodimeric hormone made of two subunits, one 92-amino-acid α-subunit and one 145-amino-acid β-subunit. Non-covalent hydrophobic and ionic interactions bind these two subunits together. While the α-subunit is common to all members of the glycoprotein hormone family [e.g. follicle stimulating hormone, thyroid stimulating hormone and luteinizing hormone (LH)], the β-subunit is responsible for the structural and functional activity of the protein [1]. The formation of an active protein requires the expression of genes encoding both subunits: the α-subunit is encoded by a single gene ubicated on chromosome 6 and the β-subunit is encoded by six allelic genes located on chromosome 19. Synthesis and assembly of hCG subunits takes place in the endoplasmic reticulum. Post-translational modifications in the endoplasmic reticulum and  Golgi apparatus lead to the production of a fully biologically active protein. Numerous functions of hCG in both pregnancy and cancer are due to glycosylation. In fact, glycosylation  patterns vary the molecular weight of hCG from 36 to 40 kDa. As a result of differences between the number, type and bonding of oligosaccharides, different variants of hCG can be found [2]. Currently, more than ten forms of hCG have been identified.  Our discussion will focus on regular or intact hCG, which is the biologically active form, and the free β-subunit (βhCG). Differentiating these forms might be useful in recognizing physiological and pathological pregnancies, as well as tumours.

Regarding its function, hCG plays a role in three different biological activities mainly related to regulation of pregnancy development and its maintenance. hCG promotes progesterone production, angiogenesis and vasculogenesis in the uterus during pregnancy, helping with the blood supply to the fetus. In addition, it seems to be one of the factors that prevents rejection of the fetoplacental issue by suppressing macrophage activity [1].

hCG: how it can be measured

We are used to seeing qualitative methods performed in urine to diagnose pregnancy, but the evaluation of the protein as a tumour marker needs more specific and sensitive methods. Indeed, protein concentrations in urine are dependent on urinary flow rate, which is one of the reasons that urine measurements of hCG can’t be used for the monitoring of cancer patients. The first commercially available hCG test identified the whole heterodimeric protein, so failed to distinguish between the different subtypes of the protein [2,3]. Fortunately, now, there are more than one hundred types of antibodies recognizing hCG-related molecules. Noncompetitive assays are commonly employed by laboratories and can be used with either polyclonal or monoclonal antibodies. In the last few years, monoclonal antibodies have improved the specificity of the assay by binding specifically to a certain form of hCG. The recognition of a single antigen results in the elimination of cross reactivity with LH (which results because they both share the same α-subunit), which was a problem with the polyclonal antibodies [1]. Particularly in the case of cancer patients, the difference between hCG subtypes is crucial. As discussed in more detail later, it has potential for recognizing both physiological and pathological pregnancies, as well as tumours.

Seminoma, a germ cell malignant tumour of the testicle
(Shutterstock.com)

The micrograph shows sheets of clear cancerous cells with a fibrous stromal network. Darker areas are chronic inflammatory infiltrates.

hCG: Utility as a biomarker in different tumours

Gestational trophoblastic diseases

The utility of the hCG as a biomarker has been widely validated in gestational trophoblastic diseases (GTD) [1]. GTD is a group of pregnancy related disorders that originate from trophoblast cells. The group consists of benign and malignant diseases, the malignant ones are known as gestational trophoblastic neoplasia (GTN), such as invasive mole, choriocarcinoma, placental site trophoblastic tumour, and epithelioid trophoblastic tumour [4].

hCG is considered the ideal tumour marker for every gestational trophoblastic disease. In fact, women with hydatidiform mole are normally monitored via hCG to detect possible persistent trophoblastic disease. According to multiple studies, sensitivity and specificity for trophoblast malignant tissue is close to 100% [1]. By measuring the circulating concentration of total hCG, it is possible to determine the amount of hydatidiform mole or tumour tissue as the relationship is proportional. This hormone is also used for diagnosis and follow-up of the effectiveness of GTN therapy. During the follow-up of these malignancies, assay of hCG alone is sufficient but the determination of βhCG can reveal a relapse earlier than hCG. The half-life of subunit free βhCG is longer, so the ratio between βhCG/hCG will increase when the levels of hCG decrease after successful therapy.

Germ cell tumours

Moreover, we can find serum hCG in germ cell tumours. Testicular cancer is the most common solid tumour malignance in males between 15–35 years of age [5]. Testicular cancers are classified as seminomas and non-seminomatous germ cell tumours (NSCGTs), the expression of hCG is different between them. Despite its lower sensitivity in testicle cancer than in trophoblastic tumours, hCG is still used in practice, as shown below. It is possible to observe the serum expression of hCG in both groups, but high serum concentrations (300–1000 IU/l) only occur in NSCGTs. Elevated serum levels of hCG occur in 40–50% of patients with NSCGT and only 15–20% in those with seminomas. The evaluation of hCG and βhCG may help to detect a relapse of the disease [1]. About the symptoms, serum hCG elevation in adult men results in gynecomastia and hyperthyroidism, whereas rising levels in children leads to early puberty.

Among female germ cell tumours, the most prevalent group is dysgerminomas [1]. These germ cell tumours of the ovary often express hCG and it can be used for daily clinical management of the tumour. As in testicular cancer, the levels of the biomarker decline after surgery or treatment, reflecting the efficacy of the treatment or operation. In addition to this, hCG levels depend on the histological type and burden of isease. When we have a histologically undifferentiated tumour, biomarkers such as hCG α-fetoprotein are a valuable clue about what type of cells form the tumour [6]. In our opinion, it’s worth mentioning that this serum marker could be useful in staging and evaluating the disease.

Other tumours

In recent years, the importance of this hormone as a biomarker of trophoblastic and non-trophoblastic tumours has increased. In fact, elevated expression of hCG in serum, urine or tumour tissue is a strong indicator of an adverse prognosis in many non-trophoblastic tumours. In particular, carcinomas of the bladder, urinary tract, lung and breast may have increased hormone levels. The immunoreactivity is mainly for βhCG (30–60%
of non-trophoblastic tumours produce βhCG but not hCG).

As mentioned before, high concentrations of hCG have been observed in human tumours that do not derive from the trophoblastic tissue. Usually, laboratories evaluate both hCG and βhCG to measure total βhCG increase. Among non-trophoblastic tumours, hCG has been described more widely in bladder and urinary tract tumours, showing a strong relationship with poor prognosis, resistance to radiotherapy and metastatic behavior. The interpretation should be cautious elevated because expression has been observed in healthy urothelial cells also [6]. In lung cancer, hCG levels in serum above 5 IU/L were observed in 12–14% of small cell lung cancers, and these were associated with short survival. Expression on serum βhCG is not associated with any specific histological subtypes, although some authors have found that is more common in adenocarcinoma [7]. With regard

to breast cancer, between 10 and 50% of patients have been found to have increased hCG in serum – this relationship has been studied but the results are not clear-cut. This finding could be related to menopausal status and cross reaction with LH: clearly elevated status is rare. However, changes in βhCG levels did not reflect properly the effectiveness of the chemotherapy.

Case report

Finally, to give a comprehensive overview of the topics discussed thus far, we are sharing our experience of a very fascinating case. A 50-year-old female non-smoker was admitted to the emergency service with stomach pain and abnormal menstrual bleeding. She had no relevant medical antecedents. The initial analysis revealed that hCG levels were elevated 3487 IU/L. Subsequent ultrasound examinations by the medical team appeared to be normal. Ultimately, she was diagnosed with extrauterine gestation versus incipient gestation. After this, treatment with methotrexate was started. A week later, the patient returned to the hospital for a routine follow-up. hCG serum levels were still elevated, so the patient was admitted to hospital. During the following days, our team discussed the case with the tumour committee, and, given her hCG levels, the decision was to do a CT scan. The imaging revealed a multi-cystic lesion in the right upper lobe of the lung, supporting the diagnosis of a malignant tumour. Our colleagues from pathological anatomy confirmed a squamous cell carcinoma of the lung (P40+ TTF1˗). The patient underwent a bilobectomy and chemotherapy. Right after these procedures her hCG levels fell (Fig. 1a). In the following year, she experienced multiple relapses, each coinciding with an elevation in hCG levels. In 2022, her last relapse, was detected initially through hCG analysis, and subsequently confirmed with the reappearance of a hypermetabolic component (Fig. 1b).

Figure 1a. Human chorionic gonadotropin levels before, during and after treatment of a multi-cystic lesion in the right upper lobe of the patient’s lung

Figure 1b. Increasing hCG levels demonstrated relapse

References
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(https://jme.bioscientifica.com/view/journals/jme/2/2/jme_2_2_004.xml).
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The authors

Rosa Ferrer*, Andrea Mansilla and Maria Costa
Department of Biochemistry, Hospital de la Santa Creu i Sant Pau, Barcelona 08041, Spain

* Corresponding author
Email: rferrerp@santpau.cat