American Journal of Otolaryngology--Head and Neck Medicine and Surgery

Lymph node micrometastasis in oral and oropharyngeal cancer. Is its clinical

significance overlooked? A narrative review
--Manuscript Draft--

Manuscript Number: YAJOT-D-23-00871

Article Type: Review Article

Keywords: Oropharyngeal Cancer; micrometastasis; isolated tumor cells; lymph nodes; neck
dissection; Immunohistochemistry

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1 Title
2
3 Lymph node micrometastasis in oral and oropharyngeal cancer. Is its clinical significance
4 overlooked? A narrative review
5
6
7 Authors
8
9 Paris Liokatisa
10 a
Department of Oral and Maxillofacial Surgery and Facial Plastic Surgery, Ludwig-Maximilians-
11
University (LMU), Munich, Germany; Paris.liokatis@med.uni-muenchen.de
12
13 Ioanna Liokatib
b
14 Department of Otorhinolaryngology, General Hospital Georgios Gennimatas, Athens, Greece;
15 Ioannaliokati@gmail.com
16
Katharina Obermeierc
17 c
18 Department of Oral and Maxillofacial Surgery and Facial Plastic Surgery, Ludwig- Maximilians -
19 University (LMU), Munich, Germany; Katharina.obermeier@med.uni-muenchen.de
20 Wenko Smolkad
21 d
Department of Oral and Maxillofacial Surgery and Facial Plastic Surgery, Ludwig- Maximilians -
22
23 University (LMU), Munich, Germany; Wenko.smolka@med.uni-muenchen.de
24 Sven Ottof
25 f
Department of Oral and Maxillofacial Surgery and Facial Plastic Surgery, Ludwig- Maximilians -
26
University (LMU), Munich, Germany; Sven.otto@med.uni-muenchen.de
27
28 Christoph Walzg
g
29 Institute of Pathology, Faculty of Medicine, Ludwig-Maximilians-University (LMU) Munich, Munich,
30 Germany; Christoph.walz@med.uni-muenchen.de
31
32
Andreas Mockh
h
33 Institute of Pathology, Faculty of Medicine, Ludwig-Maximilians-University (LMU) Munich, Munich,
34 Germany; Andreas.mock@med.uni-muenchen.de
35
36
37 Corresponding Author
38
39 Ioanna Liokati
40
Email: ioannaliokati@gmail.com
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42 Telephone: 00306945752810
43 Address: Metonos 71-73 Cholargos, Athens, Greece
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45
46
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48 Declaration of interest = None
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 1
2
3 Abstract
4
5 Background: Lymph node metastasis is considered one of the most important predictors of survival
6 in patients with oropharyngeal squamous cell carcinomas. However, the presence and clinical
7
8 significance of small tumor cell deposits in the lymph nodes have not been established yet. The aim
9 of this review is to clarify this. Metastatic findings smaller than 2 mm are considered
10 micrometastases while findings smaller than 0.2 mm are considered independent tumor cells. It is
11 unlikely that these tumor deposits can be detected by standard pathologic examination with
12
13 hematoxylin and eosin, and therefore in current practice, they do not play a role in determining the
14 patient’s decision for further treatment.
15
16 Methods: The existing studies on the clinical relevance of micrometastases in patients with
17 squamous cell carcinoma of the oropharynx were reviewed. Eleven studies that report on the clinical
18 importance of small tumor deposits regarding disease-free and overall survival, were identified.
19
20 Results: The initial results suggested that micrometastases and isolated tumor cells had no clinical
21
22
significance, but there is increasing evidence that micrometastases are also essential for prognosis in
23 head and neck oncology.
24
25 Conclusion: In contrast to other disciplines, the significance of lymph node micrometastasis has not
26 been adequately addressed in head and neck tumors. The scarcity of data and qualitative
27 prospective studies does not allow definitive conclusions to be drawn yet.
28
29
30
31 Keywords
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33 oropharyngeal cancer, micrometastasis, isolated tumor cells, lymph nodes, neck dissection,
34
35 immunohistochemistry
36
37
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39 Highlights
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41  The consensus that micrometastases do not affect survival is changing
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43  20% of the patients with OSCC have micrometastases which evade detection with H&E
44
45  A recurrence in the lymph nodes is expected in 10-15% of node-negative patients
46
47  The influence of micrometastasis on survival in patients with OSCC is still unclear
48  Current data from low quality of evidence studies are inconclusive or contradictory
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1. Introduction
4
5
Lymph node metastasis (LNM) is considered the most important predictor for survival in patients
6 with head and neck, especially oral and oropharyngeal squamous cell carcinomas (OSCC) [1]. The
7 presence of even one metastatic lymph node (LN) in the histological examination may reduce
8 survival by up to 50% [1]. Hence, lymph node metastases are often the decisive criterion for
9
10
adjuvant treatment. For this reason, the accurate classification of the N-stadium is pivotal.
11
12
The presence and clinical significance of small tumor cell aggregates in the lymph nodes is a topic
13 discussed across tumor entities. This narrative review aims to give an overview of the existing
14 knowledge regarding small tumor cell deposits in cervical lymph nodes of patients with OSCC and to
15 explore whether the fundamental question "Are micrometastases (MM) clinically significant?" can
16
17
be adequately addressed.
18
19
20
1.1 Histopathological detection of lymph node metastases
21
22 Although many technological advancements were realized in the methodology of the
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24 histopathological processing and analysis of resected tissues, hematoxylin and eosin (H&E) staining
25 remains the cornerstone of histopathological analysis. The standard protocol for histopathological
26 evaluation of small lymph nodes is a single-level sectioning, larger lymph nodes (> 5 mm) are usually
27 cut in half to enable an evaluation of 2 sections. In both cases, most lymph node volume remains
28
29 unprocessed [2]. As a consequence, it is essential to identify and dissect as many lymph nodes as
30 possible from every specimen to enable a representative estimate of the degree of regional lymph
31 node metastases. While this approach works for larger tumor infiltrates within the lymph node,
32
there is an increased probability of missing small tumor deposits, potentially leading to an
33
34 understaging of the tumor disease [3]. Serial sections combined with immunohistochemistry (IHC) or
35 PCR-based molecular techniques could improve detection sensitivity [4].
36
37
38
39 1.2 Definitions regarding lymph node metastases
40
41 The terms macro- and micrometastasis refer to the dimensions of the tumor deposits. Based on data
42 from breast tumors, the American Joint Committee on Cancer (AJCC) suggests the general cut-off
43 dimensions of 0.2 and 2.0 mm [5]. Metastatic findings smaller than 0.2 mm are considered
44
45 independent tumor cells (ITC) [6, 7], while findings larger than 2.0 mm are considered
46 macrometastases [7, 8].
47
48 Besides these quantitative measures, qualitative criteria have been proposed, too [8-10]. According
49 to them, ITC do not show signs of malignant activity (e.g., no proliferation, no stromal reaction) and
50 are located in lymphatic sinuses (no extravasation) [9, 10]. On the contrary, micrometastases are
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52 located outside the lymphatic sinus and show proliferation.
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Micrometastasis should not be confused with the term "occult metastasis", as the latter refers to
55 metastasis not detected on clinical examination and imaging [11]. Accordingly, an occult or
56 subclinical metastasis is a metastatic lymph node that was not detected by the initial imaging or the
57 clinical examination and is only diagnosed by the histological examination of the neck specimen. An
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59 occult metastasis can be both a micro- and a macrometastasis, as the sensitivity of all diagnostic
60 means is not 100%, and even macrometastases can evade them [12]. However, a micrometastasis is
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 practically impossible to be diagnosed through imaging or physical examination; consequently, a
1 detected metastasis is de facto a macrometastasis.
2
3 However, it is essential to mention that the suggested dimensions of 0.2 and 2.0 mm were based
4 mainly on practicalities and, secondarily, on data from research on breast tumors [8]. The size of 0.2
5
6 mm could be measured with a ruler and did not require special measuring devices [13].
7 Furthermore, 0.2 cm is also a size that can be reliably detected by thin sections of nodes and by
8 examining all sections with one H&E staining [13]. Nonetheless, this distinction between ITC and
9 micrometastasis can become challenging when there are multiple tumor deposits in a lymph node or
10
11 when the tumor cells are widespread [10].
12
13 Initial data suggests that micrometastases and isolated tumor cells in axillary lymph nodes from
14 breast cancers had a limited impact on survival. Therefore, additional costly procedures to detect
15 these tumor deposits might not be justified [10, 13]. In addition, it has been suggested that tumors
16 associated with an increased risk of micrometastases often have other unfavorable histopathologic
17
18 features (advanced T stage, vascular or lymphovascular invasion, etc.), which would be an indication
19 for adjuvant or systemic therapy anyway [13].
20
21
22
23 1.3 Clinical significance of micrometastases in other tumor entities
24
25 In several disciplines, the answer about the significance of micrometastases regarding prognosis is
26 becoming more apparent, and the initial consensus that they do not impact prognosis adversely
27 enough is changing. Micrometastases decrease the 5-year survival rate in patients with
28
29 adenocarcinoma of the lung from 80 to 25% [14], while their impact on survival in patients with non-
30 small cell lung cancer remains unclear [15]. Similarly, in patients with rectal cancer, lymph node
31 micrometastasis was significantly correlated with poorer disease-free survival [16]. For this reason,
32 the 8th edition of the AJCC manual announced that MM are defined as clusters of 10 to 20 tumor
33
34 cells or clumps of tumors ≥ 0.2 mm in diameter and recommended that these micrometastases
35 should be considered as standard positive nodes [17]. In oesophageal squamous cell carcinoma,
36 controlling the lymph node MM affects the prognosis [18]. On the other hand, data is contradictory
37 in patients with gastric cancer but implies that micrometastases could affect the prognosis and
38
39 recurrence rate [19]. In patients with papillary thyroid carcinoma and MM, it is unclear whether a
40 prophylactic neck dissection improves survival when micrometastases are present [20], although
41 their presence can increase the tumor marker thyroglobulin [20]. A meta-analysis from Gomez-
42 Hidalgo et al. confirmed that in patients with endometrial cancer, the presence of ITC and
43
44 micrometastases increases the recurrence rate, even if the patients receive adjuvant therapy [21].
45 Lastly, in patients with breast cancer and micrometastases in the sentinel lymph nodes, completion
46 of the neck dissection does not seem to improve survival. However, this has to be further
47 investigated for patients not receiving adjuvant radiotherapy [22].
48
49
50
51 1.4 Micrometastases in head and neck tumors
52
53 The presence of LN micrometastases in neck specimens from patients with head and neck tumors
54
55
has been studied extensively. It is generally accepted that patients with oral and oropharyngeal
56 squamous cell carcinomas classified as pN0 by the routine histological examination can still have
57 micrometastases [23]. It is estimated that approximately 20% (5-58%) of the patients have
58 micrometastases in the cervical lymph nodes, which evaded histological detection with H&E staining
59
60
[11, 23, 24]. This could explain why a recurrence in the cervical lymph nodes is expected in 10-15%
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 of node-negative patients [25, 26]. Although micrometastases seem not to be a rare finding in head
1 and neck cancers, and their presence is generally accepted [27, 28], the clinical significance of these
2 small tumor aggregates has not been adequately studied and remains unclear [11, 29]. The findings
3
4 of merely retrospective studies or small fall series are either inconclusive or contradictory [11, 30].
5 Among others, Colnot et al. investigated the bone marrow of 139 patients with head and neck
6 squamous cell carcinomas (HNSCC) using E48 RT-PCR. They reported a negative correlation between
7
micrometastases and distant metastasis-free survival [31].
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 1
2 2. Materials and Methods
3
4 In the current review, two researchers screened the literature on Pubmed, Cochrane, and Medline
5 Library until January 2023 via an electronic search query based on the terms micrometastasis,
6 micrometastases, disseminated tumor cells, oral, oropharyngeal, head and neck.
7
8 Although many articles were identified, only eleven papers reported on the clinical significance of
9
10 micrometastases in head and neck tumors. Their findings are presented in this review. The rest were
11 mainly relevant to diagnostic methods, imaging, and prognostic factors for micrometastasis or did
12 not report on clinical significance.
13
14
15
16 3. Results
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18 In an early study on this topic in 1999, Woolgar et al. [32] found that the prognosis of patients with
19 and without micrometastases is not significantly different. Woolgar et al. studied 178 patients with
20
oral/oropharyngeal squamous cell carcinoma retrospectively (table 1). They defined
21
22 micrometastases as deposits smaller than 3 mm since the above-mentioned cut-off of 2 mm was not
23 yet defined [5, 8]. They used no further methods for detecting MM other than H&E staining. The
24 data were analyzed in a univariate analysis without distinguishing between MM in pN0 and pN+
25
patients. This resulted in many patients with MM receiving adjuvant irradiation, which was not
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27 considered in the statistical analysis.
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29 In 1999 Enepekides et al. [33] studied 40 patients with oral cavity cancer by using IHC to detect CK.
30 They also found no significant correlation between MM and prognosis in the univariate analysis.
31 Similar conclusions were also reached by Kocatürk et al. in 2003 [34], who studied 22 patients with
32
supraglottic cancer by using IHC for Pan-CK.
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34 However, some later reports found a clinical correlation between micrometastasis and poor survival
35
36 by using IHC or molecular PCR-based techniques for detecting LN micrometastasis. Nieuwenhuis et
37 al. examined 41 patients in 2003 and found that patients initially classified as pN0 but with detected
38 MM by molecular techniques had worse survival than those without MM [35]. Similarly, Yamazaki et
39
al. examined 21 patients in 2005 using PCR-based mutant allele-specific amplification (MASA). They
40
41 found a worse prognosis for patients with multiple micrometastases or micrometastases in the
42 lower neck and proposed adjuvant radiation for these patients [36]. Nevertheless, none of these
43 studies examined the influence of MM in a multivariate analysis to account for other high-risk
44
parameters.
45
46 In more recent studies, Broglie et al. studied in 2012 the sentinel lymph nodes (SLN) for MM by H&E
47
48 and cytokeratin staining in 111 patients [37]. They demonstrated that even ITC could significantly
49 impact tumor control and survival in early oropharyngeal SCC in the multivariate analysis.
50 Furthermore, in 2015 Cho et al. investigated 54 LN-negative patients with OSCC [38]. They used
51
cytokeratin immunohistochemical staining to examine the LN retrospectively. The multivariate
52
53 analysis found a significant adverse correlation between survival and MM and a higher incidence of
54 MM with greater depth of invasion.
55
56 In 2018, Pu et al. examined 311 patients with OSCC using H&E staining and IHC for cytokeratin (CK)
57 [29] in a multivariate analysis. The patients with micrometastases did not benefit from radiotherapy
58 except for those with MM in levels IV/V.
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 Regarding squamous cell carcinomas in the head and neck and outside the oropharynx, Xu et al.,
1 2007, investigated 20 patients with laryngeal cancer by using immunohistochemical staining for
2 cytokeratin 19 (CK19) and loss of heterozygosity analysis [39]. They found that pN0 patients with
3
MM had significantly worse overall survival compared with pN0 patients without MM. Xu et al., in
4
5 another study in 2008, examined tumors in various sites by CK19RT-PCR and suggested that
6 detecting MM in lymph nodes of pN0 patients could be of significant prognostic value in HNSCC [40].
7 In 2012 Xu et al. further examined 126 patients with laryngeal carcinomas by cytokeratin stain [30].
8 They indicated that MM is an independent factor for poor prognosis and recommended a more
9 aggressive treatment for these patients. No multivariate analysis was performed in any of the
10
11 previous three studies.
12
13 4. Discussion
14
15 LN micrometastases from various types of tumors are being extensively discussed in the literature.
16 The initial findings suggested that micrometastases and ITC lack clinical importance [32, 33]. For this
17 reason, they were not considered for the various TNM classifications, and no additional
18
19 histopathological analysis was undertaken to detect them. However, as reported above, the standing
20 that they are not clinically significant has already changed in many oncological diseases. In contrast
21 to other disciplines, this subject is not adequately addressed in head and neck tumors, despite the
22
indications that micrometastases are also in this area essential for the prognosis.
23
24 Moreover, all relevant studies on the clinical importance of micrometastasis in head and neck
25
26 tumors were retrospectively conducted, which always comes with significant limitations.
27 Furthermore, only a few of them [27, 41] distinguish between ITC and MM by implementing the
28 lower cut-off of 0.2 mm, as defined by Hermanek et al. [8]. In addition, although LN micrometastases
29 are widely reported [24], no particular risk factors other than the already known ones for the usual
30
31 LN-macrometastases have been identified [42, 43]. More locally advanced tumors with higher
32 infiltration depths are associated with a higher risk for MM [38]. At the same time, Majumdar et al.
33 reported that even small pT1 tumors located in the tongue or the lower alveolar ridge could also
34
micrometastasize [44]
35
36 Due to the lack of reliable data that could safely conclude that micrometastases are clinically
37
38 relevant and an independent survival predictor, the standard H&E staining is not accompanied by
39 other detection methods in daily practice. This could result in an under-staging of many patients due
40 to the known limitations of H&E staining. It is clear that additional histopathological tests come with
41 an increased cost; however, in recent decades, techniques such as molecular analysis and IHC have
42
43 become widely available, and their cost has been significantly reduced. Moreover, although all
44 harvested lymph nodes in a lymphadenectomy specimen could be examined, this may not always be
45 feasible in an average laboratory. For this reason, alternative options could be considered, such as
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the examination of sentinel or high-risk lymph nodes instead of the entire neck specimen [37].
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48 The additional methods used were mainly serial sectioning with immunohistochemistry and CT-PCR.
49
50 The targets were primarily cytokeratin 19 or, alternatively, cytokeratins 5 and 14, pan-CK antibody
51 AE1/AE3 [45, 46], as well as E48, a protein responsible for the interaction of HNSCC and endothelial
52 cells [47]. Even though these methods have their own limitations, a combination of them could
53 provide the highest sensitivity and specificity, identifying patients at higher risk and preventing
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55 overtreatment in others [48].
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57 For tumor deposits in the lymph nodes smaller than 0.2 mm, data on head and neck cancers are
58 even sparser and more contradictory because, as mentioned earlier, few studies have distinguished
59 between ITC and MM and conducted multivariate analyses [46, 49, 50].
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 Concurrently, the interest in circulating tumor cells has increased in recent years, and the
1 methodology is improving [50-52]. However, it is a contradiction that the most modern methods are
2 used to identify circulating or disseminated tumor cells in various tissues, while on the other hand,
3
4 the identification of micrometastases in the lymph nodes lacks attention, although this is
5 theoretically a more straightforward task and perhaps allows more direct conclusions about
6 treatment and prognosis.
7
8
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10 5. Conclusions
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12 The scarcity of data and qualitative prospective studies regarding lymph node micrometastasis from
13 OSCC does not allow definitive conclusions to be drawn. The main question about the significance of
14
15 micrometastasis has not been convincingly answered. Once the clinical significance is demonstrated,
16 the appropriate lower threshold for micrometastasis from OSCC should be defined, since the existing
17 data originate from tumors with different histological characteristics and behavior. Similarly, the
18 most effective process of assessment, as well as the appropriate combination of cellular or
19
20 molecular targets for detecting micrometastases from OSCC has to be established. Eventually, the
21 effect of additional surgery or radiochemotherapy on small tumor deposits in OSCC remains to be
22 investigated. Prospective, high-quality studies are needed to provide definitive answers.
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 1
2
3
4 Multi- pN
Detection Number Clinically/
5 variate classification
Study Year Tumor site method other of statistically
6 Analysis of included
7 than H&E patients significant
patients
8
oral cavity/ no
19 Woolgar et al. 1999 - 178 N0,N+ no
10 oropharynx
11 oral cavity/ E48 (Ly-6D) no
2 Nieuwenhuis et al. 2003 41 N0,N+ yes
12 oropharynx RT-PCR
13 PCR-based no
14
mutant allele-
315 Yamazaki et al. 2005 oral cavity 21 N0,N+ yes
16 specific
17 amplification
18 PCR for loss of no
19 heterozygosity
20
4 Xu et al. 2007 supraglottic area (D9S171 locus) 20 N0,N+ yes
21
22 and IHC for
23 CK 19
24 yes N0,N+,
oral cavity/ IHC for
525 Broglie et al. 2012 111 Sentinel yes
26 oropharynx cytokeratin
nodes only
27
oral cavity/ IHC for yes
628 Cho et al. 2015 51 N0,N+ yes
29 oropharynx cytokeratin
30 oral cavity/ IHC for no
731 Pu et al. 2018 311 N0,N+ no
oropharynx cytokeratin
32
33 oral cavity (7)/ no
oropharynx (6)/ RT-PCR for
834 Xu et al. 2008 31 N0,N+ yes
35 hypopharynx (17)/ cytokeratin 19
36 larynx (1)
37 IHC for CK no
938 Xu et al. 2012 larynx 126 N0,N+ yes
39 (AE1/AE3)
40 IHC for CK no
10 Enepekides et al. 1999 oral cavity 40 N0 no
41 (AE1/AE3)
42 IHC for Pan- no
11
43 Kocatürk et al. 2003 larynx 22 N0 no
CK
44
45
46 Table 1. Studies reporting on the clinical significance of lymph node micrometastases in patients with head and neck squamous cell
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carcinoma
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Figure 1. Distribution of published studies reporting on the influence of micrometastasis on survival by year of publication
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 1
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