10.14639/0392-100X-N1056
Oral cancer is a heterogeneous disease that develops through a complex, multi-step process. Precision medicine should help to better understand its molecular basis, integrate traditional classifications and have a positive impact on cancer management. To apply this information in clinical practice, we need to define its histology and identify biomarkers expressed by the tumour that provide useful information for planning tailored treatment. The most reliable information currently derives from evaluation of biomarkers on post-operative samples. To plan personalised treatment, oncologists need to assess these markers on biopsy samples. We reviewed the recent literature and identified 6 of 184 publications that compared markers measured on biopsy and post-operative samples or assessed their predictivity for the development of lymph node metastases. Data from these studies suggest that markers measured on biopsy samples can provide useful indications for tailoring treatments. However, due to their heterogeneity and low level of evidence, these results need to be confirmed by clinical studies on a large population to standardise and validate biomarkers in biopsies and to assess their reliability in other diagnostic mini-invasive procedures such as radiomics and liquid biopsy.
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1056
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Mohssen Ansarin
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ACTA Otorhinolaryngologica Italica
precision medicine
Oral cancer: changing the aim of the biopsy in the age of precision medicine. A review
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2021-05-14
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Mohssen Ansarin
2021/05/14
10.14639/0392-100X-N1056
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Division of Otolaryngology and Head & Neck Surgery, European Institute of Oncology IRCCS, Milan, Italy
1827-675X
precision medicine
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Oral cancer: changing the aim of the biopsy in the age of precision medicine. A review
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ACTA OTORHINOLARYNGOLOGICA ITALICA 2021;41:108-119; doi: 10.14639/0392-100X-N1056




Review


Oral cancer: changing the aim of the biopsy in the age
of precision medicine. A review
Il cancro del cavo orale: ridefinizione del ruolo delle biopsie nell’era della medicina
di precisione. Review della letteratura
Roberto Bruschini1, Fausto Maffini2, Fausto Chiesa1, Daniela Lepanto2, Rita De Berardinis1, Francesco Chu1,
Marta Tagliabue1, Gioacchino Giugliano1, Mohssen Ansarin1
1
 Division of Otolaryngology and Head & Neck Surgery, European Institute of Oncology IRCCS, Milan, Italy; 2 Division of Pathology,
European Institute of Oncology IRCCS, Milan, Italy


SUMMARY
Oral cancer is a heterogeneous disease that develops through a complex, multi-step process.
Precision medicine should help to better understand its molecular basis, integrate traditional
classifications and have a positive impact on cancer management. To apply this information
in clinical practice, we need to define its histology and identify biomarkers expressed by the
tumour that provide useful information for planning tailored treatment. The most reliable infor-
mation currently derives from evaluation of biomarkers on post-operative samples. To plan per-
sonalised treatment, oncologists need to assess these markers on biopsy samples. We reviewed
the recent literature and identified 6 of 184 publications that compared markers measured on
biopsy and post-operative samples or assessed their predictivity for the development of lymph
node metastases. Data from these studies suggest that markers measured on biopsy samples
can provide useful indications for tailoring treatments. However, due to their heterogeneity and   Received: August 11, 2020
low level of evidence, these results need to be confirmed by clinical studies on a large popula-   Accepted: February 3, 2021
tion to standardise and validate biomarkers in biopsies and to assess their reliability in other
diagnostic mini-invasive procedures such as radiomics and liquid biopsy.                           Correspondence
                                                                                                   Francesco Chu
KEY WORDS: surgical biopsy, liquid biopsy, radiomics, depth of invasion, precision medicine        Division of Otolaryngology and Head & Neck
                                                                                                   Surgery
RIASSUNTO                                                                                          European Institute of Oncology
Il cancro orale è una malattia eterogenea che origina ed evolve con un processo complesso          via Ripamonti 435, 20141 Milano, Italy
                                                                                                   E-mail: Francesco.Chu@ieo.it
e multifasico. La medicina di precisione permette di pianificare un trattamento persona-
lizzato sulla base delle caratteristiche biologiche e molecolari delle singole neoplasie. Le
                                                                                                   Funding
informazioni oggi più affidabili sono fornite dalla valutazione post-operatoria dei biomar-
                                                                                                   None.
catori, ma per pianificare un trattamento personalizzato è necessario valutare questi mar-
catori sulla biopsia. Per questo abbiamo rivisto la letteratura dell’ultimo quinquennio ed
                                                                                                   Conflict of interest
abbiamo identificato 6/184 articoli che valutano i marcatori sulla biopsia confrontandone i
                                                                                                   The Authors declare no conflict of interest.
valori con quelli misurati sul pezzo operatorio di ciascun paziente o valutandone la predit-
tività per lo sviluppo di metastasi linfonodali. I dati che emergono da questi studi suggeri-
scono che la valutazione dei marcatori sul campione bioptico potrebbe fornire indicazioni            How to cite this article: Bruschini R, Maffini F,
utili per programmare trattamenti personalizzati. Tuttavia, a causa della eterogeneità e del         Chiesa F, et al. Oral cancer: changing the aim of
basso livello di evidenza dei lavori considerati, questi risultati devono essere confermati          the biopsy in the age of precision medicine. A re-
da studi clinici su un’ampia popolazione per standardizzare e validare i biomarcatori e la           view. Acta Otorhinolaryngol Ital 2021;41:108-
                                                                                                     119. https://doi.org/10.14639/0392-100X-N1056
loro affidabilità in altre procedure mini-invasive, ad esempio radiomica e biopsia liquida.
PAROLE CHIAVE: biopsia chirurgica, biopsia liquida, radiomica, profondità di invasione,            © Società Italiana di Otorinolaringoiatria
medicina di precisione                                                                             e Chirurgia Cervico-Facciale


                                                                                                                  OPEN ACCESS

Introduction                                                                                       This is an open access article distributed in accordance with
                                                                                                   the CC-BY-NC-ND (Creative Commons Attribution-Non-
                                                                                                   Commercial-NoDerivatives 4.0 International) license. The
Cancer of the oral cavity is the eighth most common cancer worldwide: GLO-                         article can be used by giving appropriate credit and mentio-
BOCAN reported an estimated 354,900 new cases in 2018; about 90% of these                          ning the license, but only for non-commercial purposes and
                                                                                                   only in the original version. For further information: https://
tumours are oral squamous cell carcinomas (OSCCs) with a 5-year survival of                        creativecommons.org/licenses/by-nc-nd/4.0/deed.en




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                                                      From diagnostic to predictive biopsy for tailored therapy of patients affected by oral cancer




< 60% 1. OSCCs can arise from the oral tongue, which is the         diation Therapy Oncology Group (RTOG) compared adju-
most common sub-site and has the worse prognosis 2-4, fol-          vant radiation alone vs chemoradiation in advanced head
lowed by the floor of mouth, buccal mucosa, alveolar muco-          and neck cancers. The results showed that the addition of
sa, and hard palate. Historically, OSCC affects older people,       concurrent chemotherapy was associated with better on-
mainly men, smokers and alcohol drinkers. Recent clinical           cologic outcomes compared to radiation alone in a subset
and epidemiological studies have reported an overall decline        of high risk patients. They evaluated pathological classi-
in the incidence of tobacco-related OSCCs, probably due             fication of the primary tumour, positive margins, tumour
to the anti-smoking campaigns 5-7, but an increasing trend          depth, positive nodes, extranodal extension (ENE), lym-
of tongue and oropharyngeal cancers in younger patients,            pho-vascular invasion (LVI), perineural invasion (PNI) and
women and individuals with no exposure to traditional risk          histology grade 29,30. A subsequent comparative analysis of
factors 8-10. While infection with human papillomavirus ac-         data pooled from these two trials showed that ENE and/or
counts for an increasing number of patients with oropharyn-         microscopically involved surgical margins were the only
geal cancers, risk factors, especially viruses, in OSCC non-        risk factors for which the impact of post-operative chemo-
smokers and non-drinkers, are unclear 11,12. Head and neck          radiotherapy was significant in both trials 31. Cooper et al.
cancer (HNC), including OSCC, is a heterogeneous disease            in 2012 confirmed these data by evaluating the long-term
that develops through a complex, multi-step process involv-         results of patients enrolled in the RTOG study. They found
ing genetic alterations, growth regulation, apoptosis, angio-       that at 10 years, local recurrence and disease-free survival
genesis, invasion and metastasis; moreover, it is influenced        rates were significantly better only in patients included in
by the individual’s genetic predisposition and environmen-          the chemo-radiotherapy arm for ENE and/or microscopi-
tal exposure to carcinogens 13,14. This heterogeneity is con-       cally involved margins 32. Subsequently, other studies con-
firmed in daily practice by the observation that patients with      firmed these data and proposed risk classes for effective
clinically similar tumours have different treatment responses       post-operative therapeutic planning 33-36. The 8th edition of
and outcomes 15,16.                                                 the TNM staging system 20 suggests that HPV and depth
                                                                    of invasion (DOI) as well as specific biomarkers expressed
Precision medicine                                                  by HNCs should be assessed. OSCCs are rarely related to
To address this heterogeneity, the Cancer Genome Atlas              HPV, while DOI is useful to evaluate biological behaviour,
project 17, classifications based on molecular pathology 18,        because prognosis worsens when the tumour is deeper 37,38.
and the Precision Medicine Initiative 19 have been pro-             In the past, the DOI of early-stage tongue tumours (T1-
posed. These efforts should help to better understand the           T2) was considered a predictor of occult lymph node me-
molecular basis of cancer, integrate traditional classifica-        tastases, as in skin carcinomas, and was used to plan neck
tions and have a positive impact on cancer management 20.           dissection 39,40. Before the publication of the TNM, 8th
Given this complexity, both the diagnosis and therapy of            Edition, the terms “Tumour Thickness (TT)” and “Depth
OSCCs should be planned by a highly specialised multi-              of Invasion (DOI)” were often considered synonymous.
disciplinary team that is able to globally assess the disease       However, DOI, as defined in the AJCC (American Joint
and patient 21,22. Such personalised diagnostic and treatment       Committee on Cancer) Cancer Staging Manual 8th Edition,
strategies which precisely target molecular alterations refer       has a better predictive value than TT 38,41. Recent studies
to Precision Medicine or Genomics-Driven Cancer Medi-               based on this definition suggest that DOI > 4 mm (range:
cine 23-25. To apply precision medicine in clinical practice,       3.4 mm-6.6 mm) is a strong predictor of locoregional recur-
we need to identify biomarkers expressed by tumours that            rence, and survival together with two or more markers such
reflect their individual characteristics 26-28.                     as PNI, LVI and Worst Pattern Of Invasion (WPOI-5) 42-44
                                                                    which have been integrated into the 8th TNM edition. In
Tissue biomarkers validated on post-operative surgical              addition, several studies have highlighted tumour budding
specimens                                                           (BD) and tumour-stroma ratio (TSR), considered in other
Many prognostic/predictive biomarkers have been taken               non-TNM-based staging systems, as predictive factors for
into account and tested, such as c-erbB2, EGFR (Epider-             lymphatic diffusion and disease-free survival (DFS) 15,45-47.
mal Growth Factor Receptor), tumour infiltrating lym-               Table I shows the definition and purpose of these mark-
phocytes, c-met, etc., and currently the most reliable in-          ers 2,15,37,38,42,45, 46,48-57.
formation on high-risk patients derives from assessment             To plan personalised treatment, oncologists need to assess
of tissue biomarkers on post-operative samples. In 2004,            these markers, including DOI, on biopsy samples accord-
two trials by the European Organization for Research and            ing to the AJCC TNM (8th edition) recommendations 38.
Treatment of Cancer (EORTC), and the United States Ra-              Herein, we review the literature in order to evaluate the



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R. Bruschini et al.




Table I. Pathological markers predictive of loco-regional recurrence and prognosticators.
 Marker                                                 Definition                                                            Notes
 Depth of Invasion (DOI)            DOI is measured from the level of the basement         Included in T1-3 Categories for Oral Cavity Cancer, TNM Staging
                                    membrane of the closest adjacent normal                Manual 8th Edition 37
                                    mucosa. A “plumb line” is dropped from this plane      The T category increases with every interval of 5 mm
                                    to the deepest point of tumour invasion 38,42
 Tumour Thickness (TT)              TT is measured from the surface of the tumour          As defined before publication of the 8th TNM Edition 42,48 or defined as
                                    to the deepest point of invasion. In exofitic and      an alternative to the proposed classification 49
                                    ulcerated lesions TT is measured from the              Rete pegs: epithelial extensions in the connective tissue underlying
                                    imaginary line reconstructing the intact mucosa to     the mucosa 49
                                    the deepest point of invasion 42,48
                                    TT1 is measured from the level of adjacent
                                    mucosa to the deepest point of tumour invasion 49
                                    TT2 is the distance from the bottom of most
                                    adjacent dysplastic abnormal rete pegs to the
                                    deepest point of invasion 49
                                    TT3 is measured as distance from the epithelial
                                    junction of the most adjacent dermal papillae to
                                    the deepest point of tumour invasion 49
 Radiological Depth of Invasion     rDOI is measured by drawing a perpendicular line       Radiological definition of DOI is also reported in TNM 8th Edition 37. The
 (rDOI)                             from the reference line to the deepest point of        reference line connects the junction of the tumour surfaces and of
                                    the tumour 50                                          the normal mucosa on both sides 50
 Tumour Budding (TB)                TB is defined as the presence of either isolated       TB is speculated to be the result of interactions between cancer cells
                                    single cells or small-cell clusters comprising fewer   and tumour microenvironments. It is expression of loss of cohesion
                                    than five cells scattered in the stroma ahead of       and active invasive cellular movement 55. It is considered the first step
                                    the invasive tumour front 15,45,48,51-54               in metastasis of a solid tumour
 Pattern of Invasion (POI)          POI at the tumour-host interface of oral cancer is     Tumour dispersion is assessed at the advancing tumour edge.
                                    graded 1 to 5.                                         The most common WPOI-5 phenotype is tumor dispersion
                                                                                           through soft tissue. Dispersed extratumoral peri-neural invasion,
 Worst Pattern of Invasion (WPOI)   WPOI-5 (POI Grade 5) consists of dispersed,            or extratumoral lymphovascular invasion, also can qualify for
                                    discontinuous growth pattern, with a defined           classification as WPOI-5 37
                                    tumour dispersion cutoff of 1 mm 37,54
 Tumour-Stroma Ratio (TSR)          TSR defines the interactions between cancer cells      These interactions are important for both cancer initiation and progression:
                                    and intra-tumoural stroma, which is the main           the proportion of this stroma acts as a key regulator in cancer biology and
                                    component of the microenvironment 46,56,57             could provide strategies for biological cancer treatment
 Peri Neural Invasion (PNI)         PNI is defined as the tumour cell infiltration in      PNI should be subclassified as either intratumoral or extratumoral,
                                    any layer of the nerve sheath or tumour in close       and as focal or multifocal. Extensive multifocal PNl is usually
                                    proximity involving more than one-third of the         extratumoral and frequently associated with a “strand-like” tumour
                                    nerve circumference 49                                 phenotype 37
 Lympho Vascular Invasion (LVI)     LIV is defined as the detection of tumour epithelial   LIV should be reported as either intratumoral or extratumoral, as well
                                    cells within or attached to the endothelial cell       as focal or multifocal 37
                                    lining of the vascular space 49



concordance between the values of tissue biomarkers meas-                          tumour/tumor budding. Only peer-reviewed papers were
ured in both pre-operative and post-operative samples from                         considered. We thus identified 184 papers (Fig. 1); 90 of
the same patient.                                                                  these were duplicates. The remaining 94 were evaluated
                                                                                   based on the abstracts. Inclusion criteria were: A) histo-
Methods                                                                            logically proven squamous cell carcinomas (SCC) of the
                                                                                   oral cavity; B) both prospective and retrospective studies;
For this purpose, we reviewed the recent literature pub-                           C) papers that compared biopsy and post-operative sam-
lished in English (January 2015-December 2019) by con-                             ples from the same patient; D) papers that evaluated pre-
sulting the PubMed, Medline, Web of Science, Embase                                dictivity of biomarkers on biopsy specimens; E) articles
and Cochrane databases. We used the following keywords                             based on a retrospective series whose biopsy and post-
(strings): oral biopsy and depth of infiltration/DOI; oral                         operative slides were reviewed by the authors. Exclusion
biopsy and tumour/tumor budding; oral cancer diagnosis                             criteria: a) reviews, case reports, abstracts; b) histology
and depth of infiltration/DOI; oral cancer diagnosis and                           other than SCC; c) studies that did not review histologi-



110
                                                     From diagnostic to predictive biopsy for tailored therapy of patients affected by oral cancer




Figure 1. Flow chart showing selection of papers.


cal samples. As a result of this analysis, we discarded 72         mm) in the corresponding post-operative specimens.
papers unrelated to our aim; for 12 studies, criteria for          They also evaluated TB (Tab. I) and their prognostic
patient selection or study design were not reported in de-         score (BD Model, Tab. III), and documented a sensitiv-
tail. Fourteen papers (the 10 selected articles and 4 others       ity and specificity of pre-operative vs post-operative val-
identified through a manual search) were evaluated on              ues of 59% and 100%, respectively, showing a 83% pre-
the complete text. Following this analysis, 8 more papers          operative/post-operative agreement. Seki et al. 59, among
were considered unsuitable for our purpose as they were            91 OSCC patients treated with surgery alone or preop-
reviews or the study design did not fall within the selec-         erative chemotherapy and resection, analysed 44 under-
tion criteria.                                                     going exclusive surgery. They evaluated TT, TB Score
                                                                   (TBS, Tab. III), infiltrative pattern (INF, Tab. III) and
                                                                   LVI, and found that the depth of infiltration measured on
Results
                                                                   biopsies was lower than that on post-operative samples,
All six papers included were retrospective, and were di-           while there was a good correlation of budding score in
vided into two groups based on the aim of the study                pre-treatment and post-treatment samples. Seki Soda et
(Tab. II) 48,49,58-61:                                             al. 60 recently analysed the relationship between TBS in
Comparison of tissue markers on biopsy and post-oper-              biopsies and in resected specimens of 248 patients with
ative samples. Three papers 58-60 compared the values of​​         OSCC and the effect of pre-operative chemotherapy on
biomarkers measured on patient’s biopsy and post-oper-             TBS. The authors did not report staging, but pointed out
ative samples from the same patients in order to evalu-            that 141 of 248 patients received preoperative chemo-
ate their concordance and reliability. Almangush et al. 58         therapy. The mean TBS in biopsy and resected specimens
reviewed the slides of 100 patients (81 = Stage I-II) with         showed no significant difference between patients with and
SCC of the mobile tongue treated with surgical excision.           without preoperative chemotherapy. Moreover, they found
They measured TT and found that mean values were 4.1               that pre-operative chemotherapy is effective in lowering the
mm (0.5-10 mm) in biopsy samples vs 6.3 mm (0.5-23                 budding score and suppressing relapses.



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R. Bruschini et al.




Table II. Evaluation of pathological markers on biopsy and postoperative samples in the 6 papers selected (continues on page 113)
 Author                   Study design                               Site                          No. patients               Aim of the study
                           Time period                          Stage or TNM                          Biopsy
                                                                  teatment                          technique

 Almangush,               Retrospective                      Mobile oral tongue                          100         Analysis of the sensitivity and specificity
 2018 58                   1981-2016                                                                  Incisional    of BD scores on biopsy and postoperative
                                                              Stage I-II = 81%                          biopsy                       samples
                                                             Stage III-IV = 19%
                                                              Surgery = 100%



 Seki, 2016 59            Retrospective                  Tongue, floor of the mouth                      91               Detection of histopathologic
                           2009-2013                            T1 = 43%                            Not described          parameters predictive for
                                                               T2-T4 = 57%                                          lympho nodal metastases in preoperative
                                                             Preop CT = 52%                                                   biopsy specimens
                                                              Surgery = 48%



 Seki Soda,               Retrospective                          Oral cavity                             248           Evaluation of relashionship between
 2019 60                   2009-2015                         Stage not reported                       Incisional       tumour budding score in biopsy and
                                                              Preop CT = 57%                            biopsy        resected specimens and the effect of
                                                               Surgery = 43%                                        pre-op chemotherapy on tumour budding

 Seki, 2017 48            Retrospective           Mobile tongue, buccal mucosa, palate, lip              209        Evaluation of the relashionship between
                           2009-2014                           T1-T2 = 76%                            Incisional    tumour budding in biopsy specimen and
                                                               T3-T4 = 24%                              biopsy              lymphnode metastases
                                                   Preop CT and resection +/- ND = 53%
                                                          Resection+/- ND = 47%




 Nayanar, 2019 61         Retrospective         Oral cavity (lips, buccal mucosa, tongue, hard          160                Identification of clinical and
                           2014-2016            palate, retromolar trigone, floor of the mouth)      Incision or    histopathological predictors of lymphnode
                                                                 Stage II = 48%                     wedge biopsy                    metastases
                                                                 Stage III = 20%                                       Development of a predictive scoring
                                                                 Stage IV = 32%                                                        system
                                                     Wide excision with Neck dissection
 Sahoo, 2020     49
                          Retrospective       Oral cavity (GB sulcus, tongue, floor of the mouth,      150          Comparison of prognostic performance of
                           2014-2016                        retromolar and maxilla)                  Excisional     TT and of DOI (TT2 and TT3) in predicting
                                                                     cT1/T2                           biopsy                 lymphnode metastases.
                                                    Resection and elective neck dissection                              Predictive potential of PNI and LVI



Predictive value of pathological markers in biopsy sam-                           OSCC patients (76% cT1) treated with resection or with
ples. Four papers 48,49,59,61 evaluated whether markers as-                       pre-operative chemotherapy and resection, without strati-
sessed on biopsy samples were predictive of lymph node                            fying the patients according to treatment modalities. They
metastases (Tab. II). Seki et al. 59 examined 33 patients                         found that ≥ 3 buds in the biopsy samples was predictive
with tongue and floor SCC treated with tumour resection                           of lymph node metastases, and that TBS was significantly
and neck dissection. They found that BD score ≥ 3 and                             correlated with INF, Grading and LVI (p < 0.01). The au-
TT > 3 mm was significantly predictive of nodal metasta-                          thors pointed out that the TB cut-off point needs to be de-
ses, overall survival (OS) and relapse-free survival (RFS),                       fined, and that the evaluation of TBS and TT needs to be
while TT alone measured on biopsy samples was not.                                standardised. Nayanar et al. 61 analysed 160 patients with
The same team 48 evaluated TBS on a larger series of 209                          oral cancer (Stage II = 48%) treated with wide excision



112
                                                                  From diagnostic to predictive biopsy for tailored therapy of patients affected by oral cancer




Table II. Evaluation of pathological markers on biopsy and postoperative samples in the 6 papers selected (follows from page 112)
               Markers                                                  Results                                                   Notes



                TB                                                    BD model score                              17% = non-representative biopsies (badly
                TT                                                  preo-op vs post-op                              fragmented, too superficial, technical
           BD model score                                           - Sensitivity 59.1%                                           artifacts)
                                                                   - Specificity = 100%                                      TB and TT (Tab. I)
                                                             - Agreement pre/post op = 83%                              Budding model score (Tab. II)
                                                                    Depth values (mm)
                                                            pre-op (mean) = 4.1 (0.5-10 mm)
                                                            post-op (mean) = 6.3 (0.5-23 mm)
          Histologic Grading            Tumour depth: higher in resection specimens than in biopsy samples. It                    TT (Tab. I)
                   TT                   is predictive of lymph node metastases on resection specimens, not on                    INF (Tab. I)
                  INF                                                 biopsy samples                                      Budding score (Tab. IIb)
                  LIV                   Budding score: good correlation between biopsy and postop specimens      Comparison between TB, TT, and INF status
            Budding score                BD = > 3 and Tumour depth > 3 mm significantly predictive of lymph       on lymph node metastases was assessed
                                                              node metastases, OS and RFS                         on 33 patients who underwent resection
                                             BD = > 3 and INFa/INFb: predictive of early or late lymph node                 and neck dissection
                                                             metastases or late in many cases
            Budding score                 Mean budding scores: no significant differences in both groups; but               Budding score (Tab. II)
                                                tended to increase in resected patients without preop CT
                                         Preoperative CT-S1 treatment is effective for suppressing relapse and
                                                               lowering the budding scores

                 TB                       TB => 3 predictive of Lymphnode metastases and poor prognosis              Authors describe biopsy technique;
                INF                                          P < 0.01 ( mainly T1-T2 cN0)                                      Tumour budding
            Tumour grade                 The budding score (high) together with INF and LIV were found to be     can be detected using routine haematoxylin
                 TT                       independent risk factors for lymphnode metastases at multivariate                          and
                                                                        analyses                                            eosin-stained slides or
                                         Strong correlation (p < 0.01) between the budding score and tumour               cytokeratin-stained slides.
                                                 grade, tumour depth, INF, and blood vessels invasion             Results may differ signiﬁcantly depending
                                         TB in early OSCC can be used as a reliable parameter for stratifying                on the staining used
                                                patients with different risks of lymph node metastases
         Shape of rete pegs                                         Risk score (4-18)                               The risk score needs to be tested and
          Pattern of invasion                               Score 4: predictive of N+ = 5%                          validated in other patient populations
        Depth of invasion (TT)                             Score 18: predictive of N+ = 91%                                   Rete pegs (Tab. I)
       Histologic differentiation                                                                                             Risk score (Tab. II)
            Site of cancer

          TT1, TT2, TT3, DOI                      TT2 and LVI correlate with lymph node metastases                        TT1, TT2, TT3, DOI (Tab. I)
                PNI, LVI




and neck dissection and proposed a predictive score based                       and TT3 (alternative DOI); according to the authors, TT2
on selected pathological features (rete pegs, pattern of in-                    corresponds to DOI, as defined in the TNM 8th Edition 37,38.
vasion, TT, histologic differentiation) and site of tumour                      They found that TT2 (DOI) and LVI correlate with nodal
(buccal mucosa, tongue, other). The score ranged from 4 to                      metastases.
18; the authors found that a score of 4 is predictive of risk
for lymph node metastasis of 5%; for scores of 10, 14 and                       Discussion
18, the risk was 31%, 67% and 91%, respectively. Sahoo et                       To our knowledge, this is the first comprehensive review
al. 49 studied 150 cT1-T2 patients treated with resection and                   correlating biological tumour characteristics between bi-
neck dissection for OSCC. They compared three measure-                          opsy and corresponding pathologic specimen with an ev-
ment modalities of the depth of invasion: TT1, TT2 (DOI)                        idence-based method. Unfortunately, the studies that met



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R. Bruschini et al.




Table III. Different scores for prognostic evaluation.
 Author                                            Definition                                                          Score
 Almangush, 2018 58                        BD Model Score (BDM):                                         0: < 5 buds at the IF and depth < 4 mm
                                N° of buds and depth of invasive front (IF, mm)                         1: =/> 5 buds at the IF or depth > 4 mm
                                                                                                       2: =/> 5 buds at the IF and depth > 4mm
 Seki, 2017 48                          Tumour Budding Score (TBS):                                                    Low: < 3 cells
                                             Number of buds                                                       Intermediate: 3-4 cells
                                                                                                                      High: => 5 cells
 Seki, 2017 48                             INF (Infiltrative Pattern):                     INFa: Expanding growth with a distinct border from surrounding
                                     Cancer growth and mode of invasion                                                     tissue;
                                                                                                  INFb: Intermediate pattern between INFa and INFc;
                                                                                        INFc: Infiltrative growth with no distinct border from the surrounding
                                                                                                                            tissue
 Nayanar, 2019 61                                Risk Score:                           Shape of rete pegs: Slender and fused = 1, Bulbous and uniform = 2;
                          Sum of single score of clinical and pathologic parameters                                     Irregular = 3
                                                                                          Pattern of invasion: Pushing = 1; Minimally invasive = 2; Frankly
                                                                                                                        invasive = 5
                                                                                                     Depth of invasion (TT, mm): 0-3 = 1; > 3 = 3
                                                                                              Histogic differentiation: Well = 1; Moderate = 3; Poor = 5
                                                                                             Site of cancer: Buccal mucosa = 0; Tongue = 1; Others = 2



the inclusion criteria had a level of evidence of 3 and 4                         formed peripherally to the cancer so as to obtain a signifi-
according to the AJCC on Cancer Levels of Evidence clas-                          cant amount of non-necrotic tissue from the centre of the
sification 20. It is worth stressing a criticality common to all:                 tumour 48.
namely that they differ in the design of the study, selection
of patients, oral subsite of cancer and in choice of patho-                       Depth of biopsy
logical markers such as DOI, TT and budding score; for                            It should also be stressed that the biopsy sample should be
this reason, the results are not comparable. Furthermore,                         deep enough to possibly include the entire tumour infiltra-
many authors have measured the depth of the invasion dif-                         tion front and underlying healthy tissue 48. Reliability of
ferently than what is foreseen in the TNM 8th Edition, even                       biopsy (incisional, wedge, punch, excisional) is influenced
when published after its release. In this regard, the paper of                    by the depth of the sample because it is unlikely that speci-
Driven et al. that quantified the impact of the stage migra-                      mens always include tumour stroma on the invasive front,
tion on patients’ prognosis using TT and DOI in a retro-                          which is adequate to evaluate the relationships between
spective cohort of 456 OSCC patients is interesting. They                         the various components of the tumour microenvironment
found similar stage category and prognosis regardless of                          (TME) 48,58,59. In fact, tissue biomarkers are the morpho-
the measurement used 62. This review highlights several                           logical expression of the TME, or the field in which the im-
other critical issues.                                                            mune system and the tumour interplay. Inside TME cancer
                                                                                  cells, non-tumour cells and stroma are in a continuous and
Biopsy technique                                                                  dynamic relationship, and any change within the TME may
The patients evaluated by Almangush 58, Seki 48 and Se-                           reflect changes of the balance between the immune sys-
ki-Soda 60 underwent incisional biopsy; those studied by                          tem and tumour 63. In this context, the role of biopsy is not
Nayanar 61 had an incisional or wedge biopsy; those in the                        only diagnostic, but also predictive, since biopsies should
study of Sahoo 49 were treated with an excisional biopsy.                         include the invasive front, the behaviour of tumour cells
The authors did not provide more details on the proce-                            in the TME interface and biomarkers that provide useful
dures applied to perform the incisional/excisional biopsy.                        information to guide the therapeutic choice 15,64,65-67. Leite
This information is important since the reliability of the                        et al. 66, in a recent preliminary study on biopsy samples
biopsy sample strictly depends on the quality and quan-                           from 56 OSCC cases, highlighted the possible importance
tity of the tissue taken. Seki et al. 59 did not specify the                      of the correlation between grading, predominant mode of
biopsy modalities in their first report, but in a later study                     invasion and TB intensity. The authors classified the mode
aimed to evaluate the relationship between TBS assessed                           of invasion in 4 degrees, similar to the proposal of Shimu-
in OSCC biopsy specimens and lymph node metastases                                zu 54; they also graded TB as low-intensity (5 buds in one
(LNM), recommending that incisional biopsy be per-                                single x200 power field) and high- intensity (≥ 5 buds in



114
                                                       From diagnostic to predictive biopsy for tailored therapy of patients affected by oral cancer




one single x200 power field). The majority of cases studied          Changing the aim of biopsy
(66.1%) were high-intensity TB; all cases with the worst             To plan the best treatment of a tumour, one must know its
mode of invasion showed high-intensity TB, while no as-              histopathological diagnosis, site, size and biological ag-
sociation was found between TB and histopathological                 gressiveness. Currently, the risk of developing lymph node
grading. They concluded that both TB and mode of inva-               metastases can be evaluated by measuring biomarkers such
sion in OSCC diagnostic specimens could help to select               as Grading, PNI, LVI, positive resection margins, depth and
patients who could benefit from more aggressive treatment.           mode of invasion and tumour budding on post-operative
This paper has some critical issues, such as the biopsy              specimens. On the basis of this information, high-risk pa-
performed in the central part of the tumour, which is rich           tients can be identified for treatment with post-operative ra-
in necrotic tissue that must be eliminated, and the lack of          diotherapy or chemoradiotherapy. It is hoped that this pre-
follow-up. Therefore, these promising data require confir-           dictive and prognostic information will be available for all
mation on larger series with a longer follow-up. A large             patients, even those candidates for non-surgical therapies,
biopsy on the surface (at least 8 mm) and depth (at least            at the end of the diagnostic process, so as to personalise
5 mm) could allow an assessment of the microenviron-                 the therapy for each. This review documents the possibility
ment in early cancers, but does not solve the problem 48,64.         to reliably test these tissue biomarkers on biopsy samples.
In this regard, Dhanda 64 compared TT measurement on                 However, it is necessary that the biopsy technique and the
biopsies and the corresponding post-operative samples re-            methods of evaluation of biomarkers are standardised and
ported in the medical records of 93 patients operated on for         validated in clinical trials on large case series before they
oral cancer; we have not included this paper in our review           can be used in clinical practice. For these reasons, the role
according to the exclusion criteria (point c) because those          of biopsy is changing and evaluation of a biopsy sample
specimens were not revised by a pathologist. However,                will allow not only to define the morphology of a tumour,
measurements reported in medical charts provide interest-            but also to guide the therapeutic choice.
ing information because in this series 20% of tumours had
a tumour depth > 10 mm on post-operative specimens; on               Clinical and imaging evaluation of depth of infiltration
biopsies only 28% of samples had a thickness greater than            The 8th edition of TNM suggests that the DOI of oral can-
the DOI calculated on the post-operative specimens. They             cer is assessed pre-operatively with clinical and imaging
therefore suggested performing a 10 mm deep punch bi-                investigations 38. It should be emphasised that clinical
opsy, which should potentially include the invasive front in         evaluation is subjective, depends on the experience and
up to 80% of the cases analysed in this paper 64.                    ability of each physician and is limited by anatomical
                                                                     conditions such as trismus and/or the patient’s intoler-
Additional biopsy related issues                                     ance to palpation for pain 72. Recently, prospective stud-
It should also be pointed out that 17% of biopsy samples             ies comparing pre-operative clinical and magnetic reso-
are not representative of the cancer due to technical errors         nance imaging (MRI) evaluation 72, or MRI alone 50,73,74
such as fragmented, superficial, non-orientable samples              with post-operative DOI have documented a good cor-
and artifacts in the preparation of slides 58. For this reason,      relation of these investigations, especially in tumours
how the specimen is sectioned and oriented is important:             with infiltration > 5 mm. On the contrary, other authors
slides are evaluable if sections are perpendicular and in-           have reported an overestimation of T2 lesions and under-
clude all mucous, submucosal and muscle layers. On the               estimation of T4 cancers when clinical T staging is com-
contrary, tangential sectioning does not allow the evalua-           pared with histopathological data 75,76. These papers also
tion of infiltration depth 68. In addition, biopsy and surgical      highlight several critical issues related to imaging (MRI/
procedures can sometimes make it difficult to measure the            computed tomography, CT) such as artifacts due to move-
depth of invasion on post-operative specimens. This criti-           ment and dental fillings; the evaluation of peritumoural
cal point is underlined by Berdugo who analysed the post-            oedema and inflammation, and timing between imaging
operative samples of 239 glossectomies 69: 5% had only               and biopsy (first imaging or biopsy? If imaging is per-
a minimal residual of cancer and in 14% no tumour was                formed after biopsy, how long does it take to get reliable
found. He also emphasised that in 21/183 (11.5%) pT2 OS-             information from MRI/CT?) 50,76,77. Tumour shrinkage of
CCs the DOI was underestimated due to positivity of deep             formalin-fixed post-operative specimens may interfere
resection margins. Finally, it should be kept in mind that a         with comparing the DOI between MRI pre-operative im-
large surgical biopsy may create an inflammatory reaction            aging and post-operative specimens 72,78. A recent meta-
which alters the microenvironment and could promote the              analysis 79 documented a high correlation of TT measured
regional spread of cancer 70,71.                                     by intraoral ultrasound and histopathology mainly in



                                                                                                                                              115
R. Bruschini et al.




T1-2 tongue cancers (p < 0.001), although it is operator-          also promising, although only preliminary results are cur-
dependent and often overestimates exophytic tumours 80,81.         rently available which require further confirmation. Further
                                                                   clinical studies are needed to standardise and validate clini-
Future directions                                                  cal and imaging evaluation, surgical and liquid biopsy tech-
Other non-invasive diagnostic modalities are being consid-         nique, sampling of specimens, and choice of biomarkers
ered to support evidence-based clinical decision-making, in        and their assessment before introducing these diagnostic
particular radiomics and liquid biopsy.                            and predictive modalities in daily clinical practice.
Radiomics is an emerging translational field of re-
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