Prognostic value of apoptotic index in cutaneous basal cell carcinomas of head and neck

Oral Oncology 35 (1999) 541±547

Prognostic value of apoptotic index in cutaneous basal cell carcinomas of head and neck S. Staibano a, L. Lo Muzio b,*, E. Mezza a, G. Argenziano c, L. Tornillo a, G. Pannone b, G. De Rosa a a

Department of Biomorphological and Functional Sciences, Pathology Section, Faculty of Medicine and Surgery, University ``Federico II'', Naples, Italy b Institute of Dental Sciences, Faculty of Medicine and Surgery, University ``Federico II'', Naples, Italy c Dermatologic Clinic, Precancerosis Section, Faculty of Medicine and Surgery, University ``Federico II'', Naples, Italy Received 21 December 1998; accepted 11 January 1999

Abstract Basal cell carcinoma (BCC) is the most common type of human cancer, often locally invasive, and following a benign clinical course. However, a proportion of BCCs do recur after treatment, causing extensive local tissue destruction, seldom metastasizing. Morphological methods to unequivocally distinguish the aggressive forms of these tumors (BCC2) from the ordinary ones (BCC1) have so far been lacking. Apoptosis, or programmed cell death, is thought to be important for the death of tumor cells in various stages of carcinogenesis. We analyzed the extent of apoptosis in BCCs of head and neck in a morphological, morphometric, and electron-microscopic study, to estabilish on a retrospective basis, the relative frequency of recurrence of tumors showing di€erent apoptotic rates. We found that BCC1 showed lower apoptotic index (AI) than BCC2 [BCC1: AI from 2.03 to 10.45% (mean value: 5.98%) BCC2: AI from 21.91 up to 43.82% (mean value: 39.82%)]. The morphometric analysis of both BCC1 and BCC2 revealed signi®cant di€erences between the values concerning nuclear area, length, perimeter, and roundness of the apoptotic cells with respect to the `viable' neoplastic cells. Electron-microscopy con®rmed that the features of morphological apoptotic cells were characteristic of programmed cell death. We hypothesized that low apoptotic rates in BCC1 could be indicative of a good prognosis. In fact, this corresponded to an `expansive' but not still invasive neoplastic state. In this phase, however, the tumor cells may constitute the target for genetic changes triggered by enviromental physical or chemical mutagenic agents, such as UV rays. BCC2, then, could be the result of newly selected mutated neoplastic cellular clones, with more aggressive biological behavior. The high apoptotic level found in BCC2 could thus be used as an indirect alarm signal from pathologists. This hypothesis seems to be supported by most of the current data in the literature and by the clinical outcome of BCC2 of our series. In our opinion, routine evaluation of apoptosis in BCCs could be proposed to facilitate their sub-classi®cation, contributing toward the evaluation of the prospective outcome of the individual patients. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Basal cell carcinoma; Apoptosis; Prognosis; Apoptotic index; Head and neck; Morphological study; Morphometric study; Electronmicroscopic study; Nuclear area; Length; Perimeter; Roundness

1. Introduction Basal cell carcinoma (BCC) is by far the most common type of human cancer, usually occurring in the fourth decade of life or later, as a solitary lesion on sun-exposed skin [1±4]. Individual BCCs di€er in their clinical and biologic behavior [2,5,6]. As a rule, in fact, they are low-grade tumors, only locally invasive and destructive, following a benign clinical course, but

* Corresponding author. Via Carelli 28, 71100 Foggia, Italy. Tel./fax: +39-0881-685809.

sometimes show deep local invasion and a tendency for multiple recurrences (with reported rates ranging from 0.5 up to 14%), seldom metastasizing [7]. So, although most BCCs (BCC1) present no diculty in either diagnosis or therapy, a small proportion (BCC2) do recur after treatment, eventually causing extensive local tissue destruction [8]. The appropriate management of recurrent BCCs still gives rise to considerable controversy. In fact, patients bearing the aggressive form of BCC frequently must undergo multiple operative procedures, sometimes with functional and esthetic deformities, as well as with an increased mortality rate [7,9]

1368-8375/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S1368-8375(99)00028-7

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Therefore, it seems imperative to subclassify histologically these neoplasms in order to suggest to the clinician which tumors are likely to recur, and then to require an appropriate, more aggressive treatment [9]. In view of this, several attempts have been made to determine which morphological parameters are able to unequivocally identify the lesions with a signi®cant recurrence and metastatic potential. Particularly, it has been suggested that some histological features may predict aggressive behavior, with special interest having been focused on nuclear polymorphism of neoplastic basal cells, increased mitotic index, absence of the characteristic peripheral cellular palisading, and presence of thin rows of neoplastic cells in®ltrating a strongly hyalinized desmoplastic stroma [2,7±10]. Based on these histologic ®ndings, pathologists may be able, in most cases, to suggest to the clinician which tumors are likely to recur but, unfortunately, reliable morphological methods to unequivocally distinguish the aggressive tumors from the ordinary ones have so far been lacking [2,9]. In the last few years it has emerged that cancer may be a consequence of the cell's failure to die for a suppression of the cellular death pathways [11]. In particular, increasing interest has been focused on apoptosis, or programmed, active cell death, a distinct mode of cell death which is responsible for cell loss in normal and pathologic tissues and which fundamentally di€ers from degenerative cell death (necrosis) in its morphology, biochemistry, and distribution [12]. Apoptosis represents a degradative process that irreversibly alters high order DNA and chromatin organization, and is characterized biochemically by chromatin cleavage into nucleosomal oligomers, and morphologically by cell shrinkage and chromatin condensation at the nuclear membrane (hyperchromatic nuclear fragments, condensed cytoplasm and cell fragmentation) [13]. The purpose of this study was to determine the extent of apoptosis in BCCs, by means of a morphological evaluation together with morphometric and electronmicroscopic studies, in order to estabilish, on a retrospective basis, the relative frequency of recurrence of tumors with di€erent apoptotic rates and to ascertain whether or not this ®nding has any prognostic value. 2. Materials and methods 2.1. Selection of cases Among a study population of 2201 cutaneous BCCs, examined in our laboratory from 1984 to 1994, we selected 174 cases of aggressive BCC (BCC2). As in other previous studies, the morphological criteria used to identify BCC2 were the absence of peripheral cellular palisading, nuclear pleomorphism, high mitotic index,

strongly hyalinized ®brousing stroma, and irregular pattern of invasion, with thin rows of neoplastic cells [10,14±17]. Conversely, the morphological features considered for selecting BCC1 were: uniform neoplastic basal cells with oval or elongated pale nuclei, dispersed in a ®brous stroma with an organoid pattern, and the typical peripheral cellular palisading [10,14±17]. For each case of both BCC1 and BCC2, absolute prerequisites for the selection were the complete surgical excision, a minimum follow-up time of 1 year, and the absence of family history of cutaneous cancers or of otherwise predisposing factors such as radiotherapy or genetical disorders. With these criteria, 10 BCC2 and 10 BCC1 of head and neck were selected; Table 1 shows the characteristics of the 20 patients in which apoptosis was analyzed. 2.2. Assessing apoptotic index (AI) For each case, counts of apoptotic cells and apoptotic bodies were performed on 4-mm hematoxylin and eosinstained sections from 10% neutral-bu€ered formalin, paran-embedded blocks, using a 40 objective. As mentioned above, apoptotic neoplastic cells are characterized by marked cytoplasmatic and/or nuclear shrinkage, hyperchromatic, condensed nuclear chromatin, and are often surrounded by a clear pericellular halo. Apoptotic bodies appear as dense extra- or intracellular fragments of chromatin up to 2 mm in size, with or without surrounding cytoplasm [18±21].

Table 1 Clinicopathological dataa Cases Sex Site

Age Type

Follow-up Relapses Metastases Years

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 a

M F F F F M M M F M M M F M M F F M M M

Ear Lip Nose Eyelid Forehead Eyelid Eyelid Eyelid Nose Ear Nose Ear Nose Skull Ear Nose Ear Nose Eyelid Nose

M, male; F, female.

73 67 59 39 58 62 62 77 64 67 76 60 60 68 63 78 66 53 56 50

BCC1 BCC1 BCC1 BCC1 BCC1 BCC1 BCC1 BCC1 BCC1 BCC1 BCC2 BCC2 BCC2 BCC2 BCC2 BCC2 BCC2 BCC2 BCC2 BCC2

NO NO NO NO NO NO NO NO NO NO 3 3 2 2 3 3 3 1 1 2

1 2 2 1 1

2 4 4 3 4 4 4 3 5 4 4 4 4 3 2 3 3 1 1 2

S. Staibano et al. / Oral Oncology 35 (1999) 541±547

543

For each case of BCC, 1000 neoplastic cells in about 10 ®elds were counted, and the ®nal number of tumor cells counted was expressed as the percentage of apoptotic cells among the whole neoplastic population (AI). 2.3. Morphometric analysis The morphometric analysis was performed with an image analyzer Leica Quantimet 500C-Image analysis and processing system, on the same hematoxylin and eosin sections used for the assessment of AI. A videocamera JVC TK-1280E connected to a Leitz Orthoplan light microscope was used to record the images. In each slide, 200 nuclei were measured with a 40 objective (1 pixel=0.024 mm). QWIN V01.00 software was used. The Colour Image Acquire and the Colour Detect were performed in RGB (an option that allows selection in red, green and blue). Possible artefacts and image overlapping were corrected in Binary Edit. Nuclear area, length, breadth, perimeter, roundness and aspect ratio of each nucleus were measured and the values completely automated were expressed in micrometers. The length is equivalent to the longest diameter of feret and the breadth is equivalent to the shortest. The roundness represents the form factor that gives the minimum unit value of a circle and whose formula is: Roundness ˆ

Perimeter2 4  P  Nuclear area  1:0:64

Fig. 1. Non-aggressive basal cell carcinoma (BCC1) (H&E, 250).

…1† Fig. 2. Aggressive basal cell carcinoma (BCC2) (H&E, 250).

The perimeter with regard to the angle produced by the image digitalization was recti®ed using a correction factor of 1.064. The aspect ratio results from the length and breadth ratio of the object. 2.4. Electron microscopy Transmission electron microscopy (TEM) was performed on the same formalin-®xed, paran-embedded material used for assessing the AI, in order to con®rm the presence of ultrastructural signs of apoptosis. 3. Results 3.1. Apoptotic index (AI) The AI of BCC1 neoplastic cells population ranged from 2.03 to 10.45% (mean value: 5.98%; SD: 2.52). The BCC2 subgroup showed an AI of 21.91 up to 51.48% (mean value: 39.82%; SD: 8.32). Even if both BCC1 and BCC2 groups showed a considerable range of AI among neoplastic cells, it seems evident that a marked discrepancy exists among them, with BCC1 showing very low apoptotic rates (Figs. 1

Fig. 3. Non-aggressive basal cell carcinoma (BCC1) showing only a few cells with morphological signs of apoptosis (H&E, 250).

and 3), particularly if compared with the high AI of BCC2 (Figs. 2 and 4). 3.2. Morphometric analysis The morphometric analysis of the cellular populations of both BCC1 and BCC2 bearing the morphological changes due to the apoptotic death process showed

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S. Staibano et al. / Oral Oncology 35 (1999) 541±547

Fig. 4. Aggressive basal cell carcinoma (BCC2) with high apoptotic index (AI) (H&E, 250).

Fig. 5. Aggressive basal cell carcinoma (BCC2): an apoptotic body with features of nuclear origin (TEM, 7000).

values of nuclear area, length, perimeter and roundness quite di€erent from those of the `viable' neoplastic cells. The absolute values, the means and the standard deviations of the morphometric analysis of BCC1 and BCC2 groups are reported in Tables 2 and 3.

turnover and di€erentiation, and is also prominent in the death of tumor cells in various stages of carcinogenesis [12,22±25]. Particularly, reduced apoptosis may lead to an unstable kinetics stage in tissue, favoring the expansion of total cell numbers which, in tumors, correspond to the early stage of cellular expansion [26]. Moreover, a reduction of the apoptotic process may be responsible for the preservation of genetically aberrant cells, favoring neoplastic progression [26]. Starting from these postulates, intriguing considerations arise from the analysis of apoptotic rates in BCCs of this study. As shown before, the non-aggressive group of BCC exhibited a rate of apoptotic cell death appreciably lower than that of the aggressive sub-group. Considering the major part of existing reports, this ®nding could be indicative of an `expansive' neoplastic state of BCC1, characterized by mild-to-moderate cellular proliferation (low mitotic index) with almost no, or irrelevant, programmed cell death. In contrast, in BCC2 a high cellular proliferation rate, corresponding to a high mitotic index, was accompanied by high levels of apoptosis among neoplastic cells.

3.3. Electron microscopy Condensation and fragmentation of the nuclei and the cytoplasms of individual neoplastic cells were visible expecially at the periphery of the tumoral nests. The cellular fragments (apoptotic bodies) (Fig. 5), surrounded by an electron lucent halo, were sometimes phagocytized by neighboring cells, that appeared viable. No in¯ammatory cells were observed. These morphologic features appear characteristic of programmed cell death. 4. Discussion Current knowledge indicates that active cell death seems to be the major process responsible for cell death in various physiological events, such as the adult cell Table 2 Morphometric parameters of non-aggressive basal cell carcinoma (BCC) Cases

Nuclear area

SD

Length

SD

Breadth

SD

Perimeter

SD

Roundness

SD

Aspect ratio

SD

1 2 3 4 5 6 7 8 9 10 Means Min. Max.

52.09 48.72 47.36 45.21 41.88 25.15 37.21 48.05 38.69 51.47 43.58 25.15 52.09

20.90 8.90 8.57 11.99 8.09 3.18 6.18 7.61 4.58 10.10 8.20

10.55 10.24 9.41 11.62 9.65 6.83 8.12 10.47 9.62 9.40 9.59 6.83 11.62

2.91 1.20 1.17 2.70 1.48 0.74 0.85 1.36 0.92 1.03 1.34

6.65 6.46 6.62 5.47 5.96 4.92 5.98 6.21 5.60 6.94 6.08 4.92 6.94

1.40 0.82 0.82 0.85 0.79 0.48 0.62 0.62 0.77 0.51 0.62

28.90 28.08 26.97 29.51 26.97 19.73 23.66 28.15 26.64 27.76 26.57 19.73 29.51

6.68 2.55 2.58 5.36 2.87 1.35 2.00 2.73 4.25 2.37 2.90

1.24 1.22 1.16 1.46 1.25 1.16 1.13 1.24 1.37 1.12 1.24 1.12 1.46

0.15 0.09 0.07 0.23 0.13 0.07 0.05 0.10 0.08 0.01 0.11

1.60 1.61 1.44 2.17 1.64 1.40 1.37 1.70 1.72 1.35 1.60 1.35 2.17

0.38 0.28 0.24 0.58 0.34 0.23 0.17 0.27 0.31 0.25 0.24

S. Staibano et al. / Oral Oncology 35 (1999) 541±547

545

Table 3 Morphometric parameters of aggressive basal cell carcinoma (BCC) Cases

Cells

Nuclear area

SD

Length

SD

Breadth

SD

Perimeter

SD

Roundness

SD

Aspect ratio

SD

1

Non-apoptotic cells Apoptotic cells

70.57 30.28

24.48 12.60

11.37 9.55

2.09 2.82

8.08 5.07

1.65 1.27

32.47 25.47

5.70 6.12

1.15 1.69

0.08 0.47

1.43 1.98

0.25 0.73

2

Non-apoptotic cells Apoptotic cells

52.11 27.61

16.88 7.95

9.34 9.37

1.66 1.71

7.21 4.29

1.21 0.90

27.47 23.70

4.44 3.71

1.11 1.57

0.04 0.28

1.30 2.27

0.14 0.57

3

Non-apoptotic cells Apoptotic cells

58.47 27.05

10.99 8.28

9.74 8.98

0.92 2.20

7.96 4.75

0.90 1.11

29.95 23.80

2.73 4.89

1.10 1.62

0.03 0.41

1.23 1.97

0.11 0.65

4

Non-apoptotic cells Apopotic cells

71.96 32.03

20.08 10.40

11.36 9.37

1.70 2.02

8.36 5.32

1.51 1.25

32.76 25.25

4.42 4.47

1.14 1.55

0.00 0.36

1.8 1.85

0.25 0.56

5

Non-apoptotic cells Apoptotic cells

45.38 24.71

15.09 8.76

8.97 9.25

1.65 2.61

6.48 4.25

1.18 0.99

25.99 23.71

4.23 5.71

1.14 1.77

0.06 0.52

1.40 2.25

0.23 0.69

6

Non-apoptotic cells Apoptotic cells

67.64 35.32

19.78 13.02

10.70 9.49

1.76 2.05

8.21 5.54

1.33 1.44

31.25 25.65

4.82 4.87

1.10 1.49

0.03 0.40

1.31 1.82

0.15 0.63

7

Non-apoptotic cells Apoptotic cells

67.47 27.36

22.29 10.58

10.49 9.35

1.85 2.24

8.29 4.83

1.51 1.19

31.16 24.81

5.22 5.36

1.11 1.75

0.05 0.36

1.27 2.01

0.15 0.58

8

Non-apoptotic cells Apoptotic cells

70.13 32.36

21.20 13.27

10.83 9.41

1.80 2.32

8.34 5.22

1.42 1.36

31.99 25.20

4.88 5.46

1.12 1.55

0.05 0.34

1.31 1.89

0.19 0.61

9

Non-apoptotic cells Apoptotic cells

73.28 28.17

20.37 9.85

10.97 9.40

1.58 2.37

8.96 4.92

1.20 1.47

32.91 25.07

5.48 4.63

1.11 1.91

0.02 0.48

1.23 2.75

0.13 0.71

10

Non-apoptotic cells Apoptotic cells

54.78 28.36

18.39 15.70

9.17 9.62

1.65 2.87

7.61 4.47

1.84 1.54

28.20 25.29

3.65 4.09

1.09 1.69

0.05 0.37

1.20 2.15

0.18 0.71

Means

Non-apoptotic cells Apoptotic cells

63.18 29.32

3.85 2.53

10.29 9.38

0.30 0.36

7.95 4.87

0.27 0.21

30.35 24.80

0.89 0.74

1.12 1.66

0.02 0.07

1.31 2.09

0.05 0.06

Min.

Non-apoptotic cells Apoptotic cells

45.38 24.71

8.97 8.98

6.48 4.25

25.99 23.70

1.09 1.49

1.20 1.82

Max.

Non-apoptotic cells Apoptotic cells

73.28 35.32

11.36 9.62

8.96 5.54

32.91 25.65

1.14 1.91

1.40 2.75

As is currently known, apoptosis is controlled by the growth regulatory system in the organism; it is regulated by signal transduction-coupled events, and depends on the activation of a program of gene expression in dying cells [13,27]. Particularly, the proto-oncogene bcl-2, located on chromosome 18, encodes an inner mitochondrial protein that protects cells from apoptosis [28,29]. Translocations of this gene lead to the overexpression of its product (bcl-2 protein), with the consequent inhibition of programmed cell death, providing an advantage for survival to normal and neoplastic cells [29]. Bcl-2 alone is not sucient for transformation, but the surviving cells bearing the bcl-2 translocation could constitute a target for further genetic changes, and eventually a malignant clone could be produced [29]. In a previous study (comprising most of the same BCCs of this series) we found that BCC1 showed overexpression of the bcl-2 protein, whereas BCC2 did not [17]. These ®ndings were in agreement with the wellknown ability of bcl-2 to promote cell survival even when the rate of cell proliferation is not high, as in BCC1 [30]. We then hypothesized that in these tumors bcl-2 behaved as an initial oncogene, leading to less aggressive growth of BCC1 [17].

These results appear particularly attractive, also when considering that the deregulation of bcl-2 expression suppresses the apoptotic death of cells normally following the induction of DNA damage by physical or chemical agents, such as the ionizing radiations. This may lead then to the accumulation of oncogenic mutations and to the selection of more aggressive, malignant clones [31]. Moreover, it should be considered that normally in skin exposed to mutagenic UV-radiation, apoptosis intervenes to remove damaged epidermal cells, and that BCC incidence is clearly correlated with exposure to sunlight [28,29]. At present, we think that the presence of high levels of apoptosis in the BCC2 of this study could have the opposite meaning of the bcl-2 overexpression previously found in BCC1, and that this could also be in agreement with the data on the clinical behavior of the two subgroups of tumors of our series (Table 1). In other words, the `quiescent' status of the slowly growing BCC1 could underlie a neoplastic phase in which the tumor cells (overexpressing the bcl-2 protein), remain continuously exposed to the mutagenic e€ects of many enviromental physical or chemical agents, principally to UV rays, thus constituting the target for other

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genetic changes. In this phase, among the neoplastic cells the level of apoptotic death is extremely low, with little morphological evidence of apoptosis and, practically, no apoptotic body recognizable in the whole neoplastic population. In a related immunohistochemical study, comprising most BCCs of this series, we showed an overexpression of p53 protein in aggressive BCCs [15]. As is well known, alterations in the expression of the p53 tumor suppressor gene are thought to be important events in the multistep carcinogenetic process [32]. We then concluded that the overexpression of p53 protein correlated with a dedi€erentiation process in BCCs [15]. At present, cancer is considered the result of a series of genetic changes, each potentially leading to a clonal outgrowth of cells through a selective growth advantage [33]. Even for BCCs, it has recently emerged that the follow-up of tumors over several months or years has been able to reveal that certain biological characteristics (i.e. overexpression of p53 protein) appear to occur in a signi®cant percentage of patients whose outcome is poorer than that of others [10,14±17]. In view of the current paradigms of the molecular steps in carcinogenesis, we could e€ectively hypothesize that, among the indolent cellular population of BCC1 (overexpressing bcl-2), targeted by the mutagenic e€ect of environmental agents such as UV-radiation, activation of protooncogenes and/or inactivation of tumor suppressor genes, (such as the p53), may occur, contributing to determining the aggressive behavior of individual tumors. Even if molecular pathology is providing interesting clues on the pathogenesis of BCCs, it should be underlined that the ultimate goal in diagnosis is to ®nd some `morphological' ®ndings capable of easily discriminating between BCC1 and BCC2 [10,14±17]. From this point of view, then, it could be of a great relevance to determine the morphological equivalents of these cellular events, which may allow pathologists to recognize on routine preparations the features corresponding to the biological status of neoplastic cells. In our opinion, this could be the case of the analysis of morphological evidence of the apoptotic process. In view of the results of this study, we think that the ®nding of absent or low signs of apoptosis in a BCC could be indicative of a good prognosis following the surgical removal of the lesion. By the same token, a high AI could represent an important alarm signal, constituting the epiphenomenon of a tumor with greater invasive ability probably due to a newly selected mutated neoplastic cellular clone, with more aggressive biological behavior. This hypothesis seems to be supported by the clinical outcome of BCC2 of our series (Table 1). Obviously, it must be kept in mind that the evaluation of the malignant potential of a tumor, in the absence of metastasis and/or invasion, remains exceptionally

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