Journal of Cancer Immunology & Therapy

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Research Article - Journal of Cancer Immunology & Therapy (2019) Volume 2, Issue 1

Biomineralogy of lung tumors.

Maciej Pawlikowski*

Department of Mineralogy, Petrography and Geochemistry, AGH University of Science and Technology, Kraków, Poland, Europe

*Corresponding Author:
Maciej Pawlikowski
Department of Mineralogy, Petrography and Geochemistry,
AGH University of Science and Technology,
Kraków, Poland, Europe
E-mail: [email protected]

Accepted on January 24, 2019

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Abstract

The study was devoted to determining whether cancer tissues in lungs are mineralized or not. Moreover, the author aimed to find the answer for the following question: Can mineralization of tissue lead to formation of cancerous tumors, or do cancer tissues produce substances necessary for lung mineralization? Perhaps both phenomena are possible? Studies were performed using classic mineralogical methods on thin sections of cancer tissues prepared with the use of a microtome. Obtained results confirmed the presence of trace mineralization in cancer tissues. It was represented by two types of mineralization, i.e. hidden and apparent mineralization. Both types are probably factors creating deformations of DNA and leading to formation of cancer. This hypothesis has to be confirmed by experimental studies.

Keywords

Lung tumors, Tissue mineralization.

Introduction

Lung tumors

There are many varieties of lung tumors known to Oncology (Miśkowiak 1996, Watabe 2003). Their short classification is presented below (Table 1). All obtained data were collected in Tables 2-15 and showed in Figures 1-37. Results were discussed and compared with literature.

Primary tumor
Tx Latent cancer
T0 No primary tumor
Tis Carcinoma in situ
T1 Tumor diameter ≤ 3 cm; surrounded by lung or visceral pleura,
T2 Tumor diameter >3 cm; invades visceral pleura; atelectasis or inflammation
T3 Invades the chest wall, diaphragm, parietal pleura or pericardium as well as main bronchi closer than 2 cm from the carina, but without invading the carina
T4 Invasion of mediastinum, heart, large vessels, trachea, carina, esophagus or spine, pleural effusion in the pleural cavity
Lymph node staging
N0 No lymph nodes are affected
N1 Ipsilateral bronchial or hilar nodes
N2 Ipsilateral mediastinal or subcarinal nodes
N3 Contralateral mediastinal or contralateral hilar nodes, supraclavicular nodes
Distant metastasis
M0 No distant metastasis
M1 Distant metastasis
Latent cancer Tx N0 M0
Stage 0 Tis carcinoma in situ  
Stage I T1 N0 M0
T2 N0 M0
Stage II T1 N1 M0
T2 N1 M0
Stage IIIA T3 N0 M0
T3 N1 M0
T1-3 N2 M0
Stage IIIB any T N3 M0
T4 any N M0
Stage IV any T any N M1

Table 1. TNM classification of malignant lung tumors (by Pawlicki, 1995)

                                    Element                       Content (wt%)
Na 5.5
Mg 0
Al. 0.82
I 0
Ca 8.83
Fe 0
Sn 0
P 1.58
S 20.21
K 3.74
Cl 3.75
N 0

Table 2. Results of chemical analysis of sample Figure 1.

                                Element Content (wt%)
Na 0
Mg 0
Al 0
Si 0
Ca 4.17
Fe 0
Sn 2.25
P 0
S 2.56
K 0
Cl 0
N 11.2

Table 3. Results of chemical analysis of sample Figure 2.

                                 Element Sample (wt%)
Na 0
Mg 0
Al 0
Si 0
Ca 0
Fe 0
Sn 0
P 3.65
S 4.31
K 0
Cl 0
N 20.76

Table 4. Results of chemical analysis of sample Figure 3.

                                  Element Content (wt%)
Na 0
Mg 0
Al 0
Si 0
Ca 4.34
Fe 0.67
Sn 0.59
P 3.58
S 2.89
K 0
Cl 0
N 0

Table 5. Results of chemical analysis of sample Figure 10.

                                  Element Content (wt%)
Na 0.36
Mg 0.42
Al. 0
Si 0
Ca 0
Fe 0
Sn 0
P 17.02
S 17.48
K 0
Cl 0
N 0

Table 6. Results of chemical analysis of sample Figure 11.

                                  Element Content (wt%)
Na 0
Mg 0
Al 0
Si 0
Ca 4.21
Fe 0
Sn 0.47
P 3.46
S 4.9
K 0
Cl 0
N 0

Table 7. Results of chemical analysis of sample Figure 14.

                            Element Content (wt%)
Na 0
Mg 0
Al 0.42
Si 0
Ca 0
Fe 0
Sn 0
P 12.99
S 27.06
K 10.28
Cl 0
N 0

Table 8. Results of chemical analysis of sample Figure 16.

                            Element Content (wt%)
Na 0
Mg 0.06
Al 0
I 0
Ca 0
Fe 0
Sn 0
P 31.95
S 12.3
K 4.3
Cl 0
N 0

Table 9. Results of chemical analysis of sample Figure 17.

                            Element Content (wt%)
Na 1.17
Mg 0
Al 0
Si 0
Ca 0
Fe 0
Sn 0
P 3.37
S 4.86
K 0
Cl 0
N 0

Table 10. Results of chemical analysis of sample 3 (Figure 21).

                            Element Content (wt%)
Na 0
Mg 0
Al. 0.04
Si 0
Ca 2.14
Fe 0
Sn 0.38
P 0
S 1.53
K 0
Cl 0
N 0

Table 11. Results of chemical analysis of sample 3 (Figure 22).

                            Element Content (wt%)
Na 0
Mg 0
Al. 0
Si 0
Ca 0.76
Fe 1.16
Sn 1.99
P 2.3
S 2.74
K 0
Cl 0
N 0

Table 12. Results of chemical analysis of sample 3 (Figure 23).

                            Element Content (wt%)
Na 0
Mg 0
Al. 0
Si 0
Ca 0
Fe 0
Sn 0
P 18.73
S 26.73
K 0
Cl 0
N 0

Table 13. Results of chemical analysis of sample 4 (Figure 28).

                            Element Content (wt%)
Na 0
Mg 0
Al. 0
Si 0
Ca 0.82
Fe 0
Sn 0.69
P 4.42
S 4.45
K 0
Cl 0
N 0

Table 14. Results of chemical analysis of sample 4 (Figure 29).

                            Element Content (wt%)
Na 0
Mg 0
Al. 0
Si 0
Ca 0
Fe 0
Sn 0
P 9.16
S 24.25
K 0
Cl 0
N 0

Table 15. Results of chemical analysis of sample 4 (Figure 31).

cancer-immunology-therapy-biological-microscope

Figure 1: Carcinoma planoepitheliale 1.3.1 according to WHO. Visible histological image of poorly differentiated squamous cell carcinoma infiltration. Biological microscope, magnification 40X.

cancer-immunology-therapy-bronchial-cartilage

Figure 2: Carcinoma planoepitheliale 1.3.1 according to WHO. Image of infiltration of bronchial cartilage. Biological microscope, magnification 40X.

cancer-immunology-therapy-Scanning-microscope

Figure 3: Carcinoma planoepitheliale 1.3.1 according to WHO. Selected area is enlarged in Figure 4. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-EDS-analysis

Figure 4: Carcinoma planoepitheliale 1.3.1 according to WHO. Tissue area covered by the EDS analysis. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-EDS-analysis

Figure 5: Graph of the content of elements in the test area of tumor tissue (Table 2).

cancer-immunology-therapy-tissue-image

Figure 6: Carcinoma planoepitheliale 1.3.1 according to WHO. Tumor tissue image. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-Enlarged-area

Figure 7: Carcinoma planoepitheliale 1.3.1 according to WHO. Enlarged area from Figure 6. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-test-area

Figure 8: Graph of the content of elements in the test area of tumor tissue (Table 3).

cancer-immunology-therapy-Enlarged-area

Figure 9: Graph of the content of elements in the test area of tumor tissue (Table 4).

cancer-immunology-therapy-Visible-squamous

Figure 10: Carcinoma planoepitheliale 1.3.1 according to WHO. Visible squamous cell carcinoma infiltrating the bronchial cartilage. Biological microscope, magnification 40X.

cancer-immunology-therapy-magnification-squamous

Figure 11: Carcinoma planoepitheliale 1.3.1 according to WHO. Marked area is enlarged in Figure 31. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-tumor-tissue

Figure 12: Graph of the content of elements in the test area of tumor tissue (Table 5).

cancer-immunology-therapy-magnification-content

Figure 13: Graph of the content of elements in the test area of tumor tissue (Table 6).

cancer-immunology-therapy-Mineralized-area

Figure 14: Carcinoma planoepitheliale 1.3.1 according to WHO. Mineralized area. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-Mineralized-area

Figure 15: Graph of the content of elements in the test area of tumor tissue (Table 7).

cancer-immunology-therapy-Enlarged-area

Figure 16: Differentiation of tissues in Carcinoma planoepitheliale 1.3.1 according to WHO. Biological microscope, magnification 40X.

cancer-immunology-therapy-Mineralized-area

Figure 17: Area of tested, mineralized tumor tissue, Carcinoma planoepitheliale 1.3.1 according to WHO. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-Enlarged-tested

Figure 18: Carcinoma planoepitheliale 1.3.1 according to WHO. Enlarged tested area from Figure 17. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-EDS-analysis

Figure 19: Graph of the content of elements in the test area of tumor tissue (Table 8).

cancer-immunology-therapy-EDS-analysis

Figure 20: Graph of the content of elements in the test area of tumor tissue (Table 9).

cancer-immunology-therapy-Biological-microscope

Figure 21: Carcinoma planoepitheliale 1.3.1 according to WHO. Biological microscope, magnification 40X.

cancer-immunology-therapy-bronchial-cartilage

Figure 22: Carcinoma planoepitheliale 1.3.1 according to WHO. Marked area A is enlarged in Figure 23. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-Scanning-microscope

Figure 23: Area B of mineralized tissue marked in Figure 22. Magnification according to scale.

cancer-immunology-therapy-EDS-analysis

Figure 24: Mineralized tissue structure of Carcinoma planoepitheliale 1.3.1 according to WHO. Enlarged area A. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-EDS-analysis

Figure 25: Graph of the content of elements in the test area of tumor tissue (Table 10).

cancer-immunology-therapy-tissue-image

Figure 26: Graph of the content of elements in the test area of tumor tissue (Table 11).

cancer-immunology-therapy-Enlarged-area

Figure 27: Graph of the content of elements in the test area of tumor tissue (Table 12).

cancer-immunology-therapy-test-area

Figure 28: Adenocarcinoma pulmonis 1.3.3 according to WHO. Black areas mark the dust deposits of carbon in the lymph nodes. Biological microscope, magnification 40X.

cancer-immunology-therapy-Enlarged-area

Figure 29: Preparation 124303 Sample 4, Figure 28. Adenocarcinoma pulmonis 1.3.3 according to WHO. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-magnification-content

Figure 30: Graph of the content of elements in the test area of tumor tissue (Table 13).

cancer-immunology-therapy-magnification-squamous

Figure 31: Adenocarcinoma pulmonis 1.3.3 according to WHO. Enlarged mineralized area A and B. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-bronchial-cartilage

Figure 32: Adenocarcinoma pulmonis 1.3.3 according to WHO. Area of elevated level of elements (see in Table 13). Scanning microscope, magnification according to scale.

cancer-immunology-therapy-magnification-squamous

Figure 33: Graph of the content of elements in the test area of tumor tissue (Table 14).

cancer-immunology-therapy-Enlarged-area

Figure 34: Adenocarcinoma pulmonis 1.3.3 according to WHO. Mineralized tissue. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-bronchial-cartilage

Figure 35: Structure of adenocarcinoma pulmonis 1.3.3 according to WHO. White-calcium phosphates. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-Enlarged-area

Figure 36: Adenocarcinoma pulmonis 1.3.3 according to WHO. Area A is magnified in Figure 32. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-bronchial-cartilage

Figure 37: Adenocarcinoma pulmonis 1.3.3 according to WHO. Contact of mineralized (lower) and not mineralized (upper) tissue. Scanning microscope, magnification according to scale.

cancer-immunology-therapy-Enlarged-area

Figure 38: Hypothetical DNA deformation in the section responsible for multiplication during cell division, in the environment with excessively mineralized PO43- ions (after: Pawlikowski 1995). A) Preliminary phase of DNA division. B) Division of DNA into two parts in a cellular fluid with an increased content of ions, e.g. PO43- .The ion building into the DNA in the section regulating cell procreation. C) Two new DNA spirals formed during cell division. The left helix deformed by substitution of the ion goes into a state of permanent multiplication.

Due to limited availability of the research material, this article presents the study results of only two types of cancerous tissues: squamous cell carcinoma and adenocarcinoma.

Squamous cell carcinomas are typical for smokers and constitute 40%-50% of all bronchial tumors. They exhibit features of squamous differentiation, i.e. presence of keratosis and/or intercellular bridges. They are located mainly in large bronchi, from where there is a relatively slow local growth into the environment, completely independent of the existing anatomical barriers. In each case, the tumor tissue bulges into the bronchial lumen, giving a fairly characteristic picture in bronchoscopy.

Among the subtypes of squamous cell carcinoma there are: papillary, small cell, clear cell, and basaloid. Some researchers differentiate also pleomorphic, spindle cell and papillomatous squamous cell carcinoma.

Squamous cell carcinoma gives the longest survival time without treatment, exceeding one year from the first clinical symptoms. Metastasis occurs relatively late, to peribronchial lymph nodes, tracheal carina, and then via blood vessels to other organs.

Due to the relatively slow development of the disease and late appearance of metastases, the basic therapeutic procedure is surgery. In non-operative cases, radiotherapy is used. The fiveyear survival rate in squamous cell carcinoma is around 15%, however, it largely depends on the advancement of the process [3,9,17]. Adenocarcinoma accounts for approximately 20% of all lung cancer cases. Its association with smoking is less pronounced than in squamous or small cell lung cancer.

The latest classification distinguishes the following types of adenocarcinomas: acinar, papillary, bronchioloalveolar, solid and mixed. Other rare varieties are distinguished: fetal, mucinous, mucinous cystic, signet cell and clear cell carcinoma.

Adenocarcinomas form a histologically multiform group. They are generally characterized by highly diversified pattern; however, they are quite different in terms of malice. There are tumors in which mucus production by tumor cells is present, or formation of glandular structures.

Adenocarcinomas can be located on different levels of the bronchial tree. In the main or lobar bronchi, glandular carcinomas with a very low degree of malignancy, i.e. adenomas, are generally located. They originate from glands of the bronchial wall. The main mass grows in the form of a polypoid to the bronchial lumen. This form is basically nonmetastatic and can be classified as a locally malignant tumor.

In the large bronchi, types of tumors typical for different organs may appear: cylindromatous carcinoma typical for salivary glands and mucinous epithelial tumor usually seen in salivary glands and the mucosa of the nose and paranasal sinuses.

In the distal bronchioles, adenocarcinomas with a high tendency to generalization are frequently located. They occur much more often than all the other forms in this group. They are located among the richly vascularized pulmonary parenchyma and metastasize quickly through the blood vessels.

Two subtypes of bronchioalveolar carcinoma may develop on the border of the respiratory part of the lung: mucinous and non-mucinous. These tumors are characterized by growth displacing pneumocytes on the surface of the alveoli and lining the alveolar spaces of the lungs. Broncho-alveolar carcinoma develops multifocally in a given pulmonary area. These outbreaks do not usually exceed one lobe.

Currently, there is a rapid increase in morbidity and mortality of lung adenocarcinoma, especially in women. This tumor develops peripherally and has scant symptoms. Prognosis and treatment are similar to those of squamous cell carcinoma. Five-year survival rate is determined at 19% [3,9,17].

Results

Obtained results are present below as Tables 1-15 and Figures 1-37.

Sample 1. Carcinoma planoepitheliale 1.3.1 according to WHO

Left lung Tumor, Male, Age 42.

Sample 2. Carcinoma planoepitheliale 1.3.1 according to WHO

Left lung tumor, Male, Age 59:

Sample 3. Carcinoma planoepitheliale 1.3.1 according to WHO.

Left lung tumor, Male, Age 67:

Discussion

The studies have shown two types of mineralization in the analyzed lung cancer tissues: hidden mineralization and apparent mineralization.

Hidden mineralization

Hidden mineralization was recognized for example in a sample of lung cancer (Carcinoma planoepitheliale) obtained from a 42-year-old man. It was recognized similarly to the mineralization of other specimens, by testing non-stained microtome-cut tissue sections that were deparaffinized after slicing, placed on a slide and analyzed using a scanning microscope (Jeol 540) with an EDS analysis attachment.

The sections were microtome-cut into thicker slices than required in typical histological studies, in order to avoid the influence of glass on chemical analyzes (as the cuts were placed on glass plates). The thickness of the slices used in the study was 20 μm. This is the depth to which, as verified, the electron beam of the scanning microscope does not penetrate into the tissue.

The study results show hidden mineralization in virtually all analyzed lung cancer samples. It manifests itself only in chemical analyzes, carried out by the EDS method, as elevated, abnormal contents of various elements. This applies mainly to sulfur, calcium, sodium, potassium and chlorine, but also to aluminum and phosphorus. In the same samples of cancerous tissues, spots of hidden mineralization were observed right next to spots where there was apparent mineralization and hidden mineralization in almost the same place. In many areas of cancer tissues, only hidden mineralization was observed, without visible mineral grains or crystals.

Hidden mineralization was concentrated in the examined tissues in intercellular spaces. Due to the limitations resulting from the testing method, the inside of the cells was not analyzed (the vacuum in the microscope). It can be assumed, however, that intra-cellular fluids of cancerous tissues may also show abnormal content of elements.

Apparent mineralization

This type of mineralization is definitely less frequent than hidden mineralization [10,12,13]. It manifests itself in the presence of grains ranging in size from a few to a maximum of several dozen micrometers. These grains occur separately or, less frequently, as concentrations. They usually have nongeometric, spherical or spindle shapes.

Chemical analyzes carried out using the EDS method indicate that these grains are typically composed of calcium phosphates, although those are sometimes accompanied by sulfur and other elements.

Chemical analyzes carried out using the EDS method indicate that these grains are typically composed of calcium phosphates, although those are sometimes accompanied by sulfur and other elements.

Mineral grains sometimes also locate in arterioles that nourish cancerous tissues.

Final remarks

• The tests were carried out in a very specific and unconventional way, using an electron beam for analysis of deparaffinised histological sections with a thickness of approximately 20-25 μm [7]. The obtained results prove the full usefulness of this research technique in recognizing the degree of mineralization of cancerous tissues.

• All the tested preparations presented tissues with different stages of neoplastic process and included weaker or stronger mineralization in relation to the comparative material - healthy tissues. Tissues representing the earliest stage of the neoplastic process have not been examined because at this stage neoplastic changes are often elusive.

• It was found that cancer tissue structures are less resistant to vacuum tests (scanning) than healthy tissues and are quickly destroyed under the electron beam. This may mean that cancerous tissues are mechanically less resistant than healthy tissues (as shown in Figure 38).

• In the cancerous tissues, the distribution of elements is uneven compared to healthy tissues. There are concentrations of mineral micrograins there that are mineralized with various elements in a highly variable way. This applies in particular to calcium and phosphorus, but also, to a lesser extent, sulfur and others [11].

• In cancerous tissues there is usually more calcium and potassium than in healthy tissues. On the other hand, magnesium and sometimes sodium are almost completely lacking in comparison to the amount of control group observed in the samples.

• The degree of hidden mineralization of cancerous tissues is, according to research, less dependent on the patient's age, and more on the advancement of the neoplastic process.

• In the first phase, mineralization of the area of malignant tissues always has the character of hidden mineralization [13] and is not manifested by the presence of grains but is only visible in chemical analyses as elevated amounts of elements in tissues. This means that elements such as calcium, phosphorus and sulphur build into the biological structures of tissues and do not form grains that stand out, for example, microscopically.

• Hidden mineralization can, but does not have to, turn into apparent mineralization visible as micro grain concentrations. These grains, up to several dozen micrometres in size, usually locate near cells and arterioles. Observations of calcium and phosphorus correlation indicate that mineral grains are represented by either calcium phosphates or, more rarely, calcium carbonates.

Conclusion

In conclusion, one can hypothesize that both hidden and apparent tissue mineralization, including the so-called calcification, may promote structural DNA defects arising during the division of chromosomes in the process of cell multiplication. The cell division environment with an abnormal content of elements (too low or too high mineralization) can promote deformations of the DNA segment which is responsible for the rate of mitotic divisions. Thus, the mineralization of tissues and body fluids (caused by both external and internal factors) may promote the formation of tumors.

Confirmation of this theory requires further research, including experimental research; this research and publication are dedicated to my mother, who died of lung cancer.

References