Pattern-Corrected Mitotic Activity Index (PMAI): A Novel Prognosticator of Oral Squamous Cell Carcinoma
Nunna Sai CHITRA, Karen BOAZ, Srikant N, Amitha J LEWIS, Sneha K.S.
Department of Oral Pathology and Microbiology, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, KARNATAKA, INDIA
Keywords: Oral cancer, Mitotic index, Prognosis, Survival, Squamous cell carcinoma
The main aim was to assess the efficiency of the Mitotic Activity Index (MAI) and a novel index devised by us, the Pattern-Corrected
Mitotic Activity Index (PMAI) in prognostication of Oral Squamous Cell Carcinoma in terms of lymph node involvement, margin, recurrence
and survival status.
Material and Method: The study group consisted of 60 cases of histologically-proven Oral Squamous Cell Carcinoma with known status of
prognostic indicators. Hematoxylin and eosin stained sections of the tumor proper were utilized for assessment of mitotic activity and pattern of
invasion. The Mitotic Activity Index and Pattern-Corrected Mitotic Activity Index were then calculated and correlated with the prognosticators.
Results: Mitotic Activity Index was higher in patients who had better survival and low recurrence rates. Pattern-Corrected Mitotic Activity Index
showed the greatest percentage increase in relation to lymph node involvement as compared to the other indices. Kaplan Meier survival analysis
showed that a higher Pattern-Corrected Mitotic Activity Index (>1.45) was associated with poorer survival (37.19 months).
Conclusion: Lack of significant association of the Mitotic Activity Index in relation to prognosticators could be attributed to a tumor having
a migratory phenotype rather than a proliferative phenotype as seen in late-stage tumors. Late-stage tumors have more of a poorer pattern of
invasion which is reflected best by Pattern-Corrected Mitotic Activity Index by correlating with poorer survival and lymph node involvement.
Head and neck malignancies constitute the majority of
cancers among the Indian population. The prevalence of
Oral Squamous Cell Carcinoma (OSCC) is increasing
over the last decades, with a high incidence of associated
morbidity and mortality. Despite enormous advancements
in the field of diagnostics and therapeutics, the overall
survival rate in most countries has not shown significant
improvement. The prognosis remains quite unclear as
the involvement of lymph nodes and surgical margins by
tumor directly impacts on recurrence and survival.
TNM staging and histological grading systems have been
used commonly to prognosticate OSCC (1). Bryne M. et
al. evaluated five histological features namely degree of
keratinization, nuclear pleomorphism, lymphoplasmacytic
infiltrate, the pattern of invasion and mitotic activity to
grade the carcinoma at the Invasive Tumor Front (ITF),
which is popularly regarded as a reliable prognosticator of
OSCC. Studies later have assessed individual histological
parameters to determine their efficiency in prognostication
of OSCC. The assessment of cellular proliferation is one such parameter used not only for primary diagnostic purposes
but also as a guide to prognosis. Several sophisticated
techniques are available for assessing proliferative activity
of tumors; however, the oldest, easiest, fastest and cheapest
way of assessing proliferation is by counting the number
of mitoses in tissue sections. Various histological grading
systems have incorporated mitotic activity and pattern
of invasion in their assessment with the earliest grading
systems having a subjective evaluation of mitosis whereas,
the later systems having an objective way of assessment
of mitosis. The assessment of mitotic activity is one of the
cornerstones for the diagnosis and grading of OSCC, soft
tissue sarcomas and other malignancies. Mitotic activity
index (MAI) is the oldest way of measuring proliferative
activity. It is cost effective as mitoses are counted in
routinely fixed and processed hematoxylin and eosin
(H&E) sections and if performed carefully can give very
useful information (2).
The pattern of invasion at the ITF in the form of individual
cells or strands is known to be an adverse prognosticator of
OSCC. The loss of adhesive proteins results in a dissociative pattern of invasion visualized histologically as individual
cells and strands. We hypothesized that the tumors showing
the worse pattern of invasion (like permeating as individual
cells and thin strands) having concomitantly higher mitotic
activity would have a poorer prognosis. Thus, we hereby
propose a novel mitotic index that combines the two
parameters as the Pattern-Corrected Mitotic Activity Index
Therefore, this study intended to objectively and
comparatively assess mitotic activity along with a new
proposed Pattern-Corrected Mitotic Activity Index, in an
attempt to predict the prognosis of OSCC.
Formalin-fixed, paraffin-embedded tissue blocks of 60
cases of OSCC were retrieved from the archives of the
Department of Oral Pathology, MCODS, Mangalore.
Relevant patient data (TNM staging, recurrence, and
survival) was accessed from the medical records file of the
patient. The histopathological diagnosis of margins and the
lymph node status were assessed by retrieving the slides and
histopathological reports. Patients who underwent curative
excision as the primary mode of treatment were included
in our study. The study commenced after approval from the
Institutional Ethics committee.
Sections of 4μm thickness were taken from the
representative paraffin-embedded tissue blocks and were
subjected to routine H&E staining. The archived slides were
analyzed in correlation with the grossing sheet, to identify
the invasive tumor front of the OSCC. Brynes’s grading
system was employed at the Invasive Tumor Front and the
number of mitotic figures and the pattern of invasion were
assessed for each case. The tumor was assessed in 10 HPF.
A 100-point square grid reticule (Olympus, 190cm 10x10
square) was placed in the eye-piece of the Olympus CH20i
microscope. The tumor cells were assessed at the invasive
tumor front with the grid superimposed on the field of
view. Using the grid as a reference, the number of mitotic
figures were counted in the given fields.
The number of mitotic figures was assessed in the
corresponding fields as per the criteria proposed by van
Diest et al. (3). The cell under consideration should not
have a nuclear membrane with the nuclear material having
clear ‘hairy’ extensions (condensed chromosomes) present
either clotted together (beginning metaphase), in a plane
(metaphase/anaphase) or in separate clots (telophase).
Structures and figures not satisfactorily meeting the abovementioned
criteria were excluded from the assessment.
The pattern of invasion score was assessed in each field
using Bryne’s Grading criteria where a score of 1 was given
for pushing, well-delineated infiltrating borders, score
2 for infiltrating, solid cords, bands, and strands, score 3
for smaller groups of cells having >15 cells and score 4 for
tumor showing marked widespread cellular dissociation in
small groups (n<15) or in individual cells (2).
Following the counting of mitotic figures in 10 high power
fields, and pattern of invasion the following mitotic indices
were employed. The PMAI has been newly derived by us
as a potential prognosticator as it combines the pattern of
invasion and mitotic activity of the tumor.
• MAI (Mitotic Activity Index) was defined as the number
of mitotic figures in 10 HPFs.
• PMAI (Pattern-corrected Mitotic Activity Index) was
calculated as the average of the product of the mitotic
figures and pattern score in ten fields.
Demographic data for 60 cases were summarized using
descriptive analysis. Prognostication of OSCC was
done using the parameters of pathological lymph node
involvement, margin status, recurrence and survival of
the patient. The association of prognosticators with TNM
staging was done using the chi-square test. MAI and PMAI
were compared between the better and the worse prognosis
of the above parameters using independent student t-test.
The mitotic indices were compared with the four stages of
the TNM staging system using the Kruskal Wallis test. ROC
curve analysis was performed to assess the area under the
curve and predictive efficiency (for a given cut-off) of each
mitotic index in relation to the prognosticators. The cut-off
for each mitotic index was arrived at using the coordinates
having the highest sensitivity and specificity for the event of
lymph node involvement. Using the cut-offs so derived, the
sensitivity, specificity, positive predictive value, negative
predictive value and diagnostic accuracy was calculated for
each prognostic parameter.
Kaplan Meier Survival analysis was performed for 60
patients and a comparison of the mean age of survival
was performed at the derived cut-off of each mitotic index
using the Log-Rank test.
In a cohort of 60 cases of OSCC, we have observed the
predominance of males (81.7%) with a varying age group
of 34 to 80 years and a mean age of 57.5±11.13 years. Most
of the cohorts were placed in TNM Stages IV and III (58.3% and 26.7% respectively). Brynes’ histological grading system
was used to evaluate the predominant pattern of invasion
as well as the worst pattern of invasion. Lymph nodes were
free of tumor in 73.3% of the cases and surgically resected
margins were found to be involved in two-thirds of the
cases (65.0%). Also, 55.0% of OSCC patients showed no
recurrence and the survival rate of the cohort was 75.0%
Mitotic indices like Mitotic Activity Index (MAI) and
Pattern-corrected Mitotic Activity Index (PMAI) were
compared with the prognostic parameters like lymph node
involvement, margin recurrence, and survival. Patterncorrected
Mitotic Activity Index and Mitotic Activity
Index were higher in cases with lymph node involvement
showing 49.01% and 20.38% change respectively. In the
cases showing the recurrence of tumor, poor survival and
positive surgical margins it was noted that the mitotic
indices were lower (Table II).
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|Table II: Comparison of The MAI and PMAI between the prognostýc factors using independent student T test.
MAI and PMAI were compared with TNM staging using
the Kruskal Wallis test and were seen to show the highest
median scores in stage IV tumors. The Mitotic Activity
Index showed a gradual increase from stage I through stage
IV and PMAI showed higher scores in stage I with a lower
score in stage II which increased in stage III and stage IV
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|Figure 1: Median scores
of Mitotic indices in
relation to the TNM
staging system (Box and
Receiver Operator Curve (ROC) Characteristics was
performed for each of the prognosticators with the mitotic
activity indices. The area under the curve was more than
0.5 only in the prediction of lymph node involvement,
indicating that mitotic activity was correlating with the lymph node involvement and not with margin, recurrence
or survival in our cases. Among the mitotic indices, the
higher area under the curve was shown by PMAI (0.663)
compared to MAI (0.608). Using the coordinates of the
curve based on the ROC curve for prediction of recurrence,
we arrived at an optimum cut-off of 7.5 for MAI and 1.45
for PMAI showing similar sensitivity/specificity of 62.50%
Kaplan Meier survival analysis showed that MAI had better
survival with higher values of the index. Patients with MAI
>7.5 had a mean survival rate of 92.33 months compared
to 58.98 months in patients with scores <7.5. However, the
Pattern-Corrected Mitotic Activity Index had an inverse
association. Patients with PMAI >1.45 showed poorer
survival of 37.19 months as compared to the ones having
<=1.45 PMAI with a mean survival of 73.02 months (Table
III) (Figure 2,3).
Binary logistic regression analysis shows that an involved
margin status has the highest odds of death with odds ratio
of 5.961. This is followed by a pattern-corrected mitotic
index which shows odds of 3.331 toward death when the
PMAI is <=1.45. Buccal mucosa involvement showed
higher odds of 2.009 compared to alveolus involvement in
event of death (Table IV).
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|Table IV: Binary logistic regression analysis (enter method) for assessment of the odds of survival.
Proliferation is one of the fundamental processes in
neoplasia as it plays a critical role in the growth and
sustenance of tumor. Tumor volume maintenance needs
multiplication of cells that in turn requires the process of mitosis. Proliferation markers have been utilized by many
researchers in a bid to predict prognosis. The simplest
method of assessing the proliferation is by evaluation
of the number of mitosis which can be readily identified
by routine H&E staining. The clinical TNM staging and
histological grading (Bryne’s Invasive tumor front grading
system) have been the standards in the prediction of
prognosis. One of the components of TNM staging is the
size of the tumor which characterizes the tumor load or
the quantity of the tumor along with the nodal status and
metastatic character (4).
Bryne’s grading system has included mitotic figures along
with other histological characteristics like maturation,
pleomorphism, host response and pattern of invasion and
the grading system has yielded excellent correlation with
prognosis. From amongst the other parameters of Bryne’s
grading system, the ‘pattern of invasion’ may be regarded
as an indicator of the migratory potential of the tumor.
Fundamental to neoplasia is the requirement of cells to
multiply and go forth to spread and metastasize (5).
Studies have shown that the pattern of invasion and mitotic
activity are the two most important prognosticators (6,7).
We developed the Pattern-corrected Mitotic Activity Index (PMAI) to validate our assumption that tumors with high
proliferation and greater migratory potential would have
a poorer prognosis. The novel ‘Pattern-corrected Mitotic
Activity Index’ (PMAI) quantifies the combination of the
mitotic activity and the pattern of tumor invasion at the
invasive tumor front.
In our study, MAI was correlated with prognostic
parameters and was seen to be higher in patients who
had better survival rates and low recurrence rates. These
results were similar to those reported by Davies et al.
who immunohistochemically assessed the proliferation
rate of oral squamous cell carcinoma using Ki-67 and
found a higher recurrence rate to be associated with
lower proliferation index (8). Veronica A et al. have also
reported higher survival rates correlating with higher rates
of AgNOR’s and Ki67, in oral squamous cell carcinoma of
a cohort in Uruguay (5).
Survival in patients with OSCC depends on multiple
factors. Dissanayaka et al. assessed a cohort of 193 patients
with OSCC and found a significant association between
the pattern of invasion of tumor and nodal metastases.
They reported that patients with tumors invading in
individual cells and thin strands showed a higher tendency for metastasis with poorer survival (mean of 2.7 years) as
compared to the patients with tumor invasion islands or as
broad pushing margins (mean survival of 3.7 years). Thus a
tumor having a low mitotic activity but a worse pattern of
invasion would correlate with poorer survival owing to the
higher metastatic potential of the dissemination of tumor
cells. This is demonstrated by the PMAI index efficiently
(9). The PMAI index has the ability to quantify the two
significant prognosticators of OSCC, namely, proliferation
(mitosis) and potentially migratory component (pattern
of invasion) in a unified single index. This novel index
(PMAI) proved to be an efficient prognosticator in our
cohort as it showed a positive correlation with mortality.
Patients with a higher PMAI score of >1.45 had a lower
mean survival rate of 37.19 months as compared to the
ones with PMAI<=1.45 who survived for nearly twice as
long (73.02 months) (Table III).
Lymph node (N) status is known to be the most important
clinical parameter determining survival. PMAI showed a
greater percentage increase of 49.01% in relation to lymph
node involvement as compared to the Mitotic Activity
Our lack of observed significant correlation between
nodal status and recurrence, margin and survival could
be attributed to a bias in the cases in our cohort wherein
most patients were in late-stage tumors constituting, TNM
stages III and IV owing to the nodal status being either N1
or N2 in 54/60 cases. In late-stage tumors the neoplastic
cells are more likely to have acquired a mesenchymal
phenotype that supports migration. In what could be
simplistically described as a ‘change of priorities’, the tumor
that previously focused on volume has now changed track
towards metastasis and to achieve this change in focus,
the resultant epithelial-mesenchymal transformation that
involves reorganization of cytoskeleton has since rendered
the cell incompatible for significant proliferation to occur
This phenomenon is supported by studies by Bettendorf
and Herrmann who indicated that the tumor growth is
exponential at its onset whereas in later stages the cell cycle
slows down. This was assessed by immunohistochemical
markers like Ki67, PCNA, and P34, which indicate the
increasing DNA synthetic activity in the cell, which is
higher in the early stage tumors (10). Freudlsperger et al.
found that Ki-67 labeled proliferation positively correlated
with increasing frequency of loco-regional recurrence in
stage I tumors while tumors in Stages III and IV in their
cohort showed a lack of correlation of proliferation with
survival or loco-regional recurrence. This is in agreement with our findings as most of the OSCC cases in our cohort
were in stages III and IV suggesting that the tumor has
ceased to be in active proliferation mode and is progressing
towards acquiring phenotypical changes in preparation
for epithelial-mesenchymal transformation and eventual
It is understood that the worsening TNM stage increases
the potential for metastasis as is evident by the shift in
the pattern of the tumor phenotype. After a threshold in
tumor size is achieved, the tumor environment is no longer
conducive for proliferation. This initiates the phenotypic
switching indicated by a reduction in the proliferation
markers and an increase in factors associated with the
migration of tumor cells. Thus, in late-stage tumors, one
can expect lower proliferation rates as we did find upon
correlation with mitotic indices.
Lack of a significant correlation of the mitotic indices
with prognosis could also be attributed to other factors
governing tumor biology.
It may be the quality and not merely the number of mitoses
that may correlate with the prognosis. The presence of
tripolar, tetrapolar, and atypical anaphase mitotic figures
indicate the presence of genetic aberrations and correlate
The presence of an increased number of mitotic figures may
just be related to the increased response to growth factors
rather than conferring aggression on the tumor.
As the number of cell divisions increase, tumor cells
progressively acquire and accumulate genetic aberrations
that may propel the tumor cells towards apoptosis or
migration. As explained earlier, early-stage tumors are
characterized by mitotic activity and tumor cells in the later
stages are more inclined to migrate and metastasize rather
than divide. Thus, mitotically active/dividing cells will
correlate with the size of the tumor but not the metastasis,
margin, recurrence or survival status of the individual.
In conclusion, the efficacy of mitotic indices that assess
mitotic activity alone or in conjunction with tumor volume
is questionable in predicting prognosis in late-stage
tumors. Late-stage tumors acquire a migratory phenotype
that thrives less on proliferation through mitosis and more
on the ability to metastasize. Therefore, the PMAI offers
the prospect of being a better predictor of survival as it
incorporates assessment of mitosis along with the pattern
of tumor cell invasion.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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