Pathological Society abstract
Developing microRNAs as biomarkers for cutaneous melanoma
Prashant Shah
INTRODUCTION: Cutaneous melanoma is an aggressive disease. Currently, the
2009 American Joint Committee on Cancer (AJCC) staging system is used for
malignant melanoma. Clinical and histological features act as indicators of
survival. Accurate staging following excision biopsy is vital to ensure appropriate
management. Features used for AJCC staging are Breslow’s thickness, ulceration,
mitotic rate (stage I/II), lymph node metastases (stage III), site of distant
metastases and serum LDH (stage IV). The stage of melanoma at diagnosis
strongly relates to prognosis.
Several clinico-pathological variables affect prognosis including AJCC features as
well as age, gender, anatomical site, subtype and Clark’s level. Breslow’s
thickness is established as the single most valuable prognostic guide. Patients
with thin lesions less than 1mm thick have a 5-year survival rate greater than
95%. This falls to less than 50% for tumours greater than 4mm. Metastatic
spread can occur early in development, with appalling outcome in patients.
Metastatic tumours have a 5-year survival rate ranging from 5-22%. This
highlights the necessity to detect melanoma at an early stage. The staging
protocol used at present is not supported by any molecular tests of analogous
value.
Despite the general reliability of AJCC staging in predicting outcome, limitations
exist. Some individuals with several poor prognostic factors can still survive for a
long time without any metastases, yet patients with thin primary cutaneous
melanoma can develop metastases and die from their disease. Thus, a proportion
of people exist in whom prognosis cannot be accurately determined using
clinico-pathological features.
The unpredictable behaviour of melanomas that are morphologically similar
makes prognostication for histopathologists very difficult. The clinical
application of biomarkers known to reliably act as independent prognostic
variables would be desirable. Using PCR, miR-10b has been identified as a
purported prognostic biomarker in our lab. The primary aims of this study were
firstly to validate a method for miR-10b detection. Secondly, miR-10b levels
were determined on a melanoma cohort using this method. Finally, miR-10b was
evaluated as a biomarker to support our earlier lab findings.
METHODS: 8 breast cancer and 5 melanoma cell lines were surveyed for miR10b controls by RNA extraction and real-time quantitative reverse-transcription
PCR (q-RT-PCR). Cell line controls with highest, intermediate and lowest miR10b levels were selected for pre-designed miR-10b Colorimetric In-Situ
Hybridisation (C-ISH) probe assay validation and C-ISH on melanoma cases. Cell
lines were cultured according to standard cell culture methods. C-ISH results in
cytoblock section controls were compared against q-RT-PCR data. Similar miR10b levels were expected from both techniques.
Melanoma tissue from patients with primary cutaneous malignant melanoma
was used in this study. All cases were FFPE tissue that had been used for
pathology reports. Histology specimens were obtained from the University
Pathological Society abstract
Hospital Leicester NHS Trust’s Histopathology diagnostic archive, diagnosed
before 2004-5. C-ISH evaluated the clinico-pathological significance of miR-10b
against outcome measures in 107 melanoma cases and H scores quantified miR10b. The independent variable was miR-10b expression. The dependent variable
was patient outcome, which was defined as time to first metastases. All-cause
death time and melanoma-specific death time were measured for survival
analyses. Kaplan-Meier plots and Cox proportional hazard regression analysis
determined the prognostic value of miR-10b and other variables in melanoma. A
relationship between miR-10b expression and these clinico-pathological
variables was also determined.
RESULTS: MCF7 (RQ=1), MDA-MB-231 (RQ=44) and SK-MEL-28 (RQ=753) were
used as miR-10b cell line controls based on their Relative Quantification (RQ)
values. There was a general drop in detection of miR-10b between fresh cells and
fixed cytoblock tissue. After troubleshooting, C-ISH results corresponded to qRT-PCR data in the majority of controls, thus validating the probe assay.
Preliminary C-ISH runs showed systematic bias in methodology, likely due to
technical variation. Attempts to alleviate this still left slight systematic bias
between repeat C-ISH runs on the same cases over 4 different days with an
Intraclass correlation coefficient of 0.51, indicating moderate agreement.
After proceeding to melanoma cases, H scores were allocated to 105/107 cases
with high and low miR-10b expression groups formed around a median score of
100. 2 sections were not scored due to overdigestion with Proteinase K. The
same 15 sections were scored twice on separate days to check reliability in the
operator’s scoring. The ICC value was 0.92 suggesting very good agreement in H
scoring.
Univariate analysis indicated miR-10b was not significantly predictive of patient
outcome (p>0.05). Multivariate analysis showed Breslow thickness
independently predicted all outcome measures (p<0.05). Ulceration
independently predicted time to all-cause death (p=0.02) and melanoma-specific
death (p=0.021). MiR-10b was not related to known powerful prognostic
variables.
Reasons behind the lack of significance of miR-10b on outcome effects were
sought. Batch variation was present, F(10,94)=2.047, p=0.037. However, this was
not due to inherent batch bias because there was no significant difference in
independent outcome predictors, Breslow thickness (p=0.095) and ulceration
(p=0.219) between batches. The batch variation was therefore presumably due
to technical differences from C-ISH technique or H scoring. During analysis, some
cases were difficult to score because of invasive parts of tumour section that
were separated from the main tumour, thus producing different staining
intensities.
CONCLUSIONS: The miR-10b C-ISH probe assay was validated and deemed
suitable for melanoma case application. Diminishing C-ISH signal was seen from SKMEL-28, MDA-MB-231 through to MCF7 in the majority of cytoblock sections.
However, a slight discrepancy still remained.
Our study showed miR-10b was not associated with outcome after analysis of
105/107 cases. This was most likely due to technical issues from personal C-ISH
technique or cases that were difficult to score. The fact that Breslow thickness and
Pathological Society abstract
ulceration significantly predicted outcome showed us that our cohort was
representative of melanoma. No relationship between miR-10b and other prognostic
variables further supported our findings of miR-10b having no significant effect on
outcome. If miR-10b was an important marker, it would have related to these
variables.
The lack of outcome effects was most probably due to intra-operator variability in CISH technique or case scoring. Genuine biological difference causing these effects
were unlikely because the previous study used a robust RT-PCR technique on 2
independent cohorts and screened more than 300 miRNA’s to identify miR-10b as a
sole, independent predictor of outcome. Measures were taken to minimise technical
error by reducing batch variation in preliminary C-ISH runs, but systematic
differences persisted. Due to time constraints, it was deemed suitable to proceed to CISH analysis of cases, keeping in mind technical variation being responsible for any
outcome effects. The lack of support in our study in comparison to the previous study
indicates further research must be performed to mitigate technical error and confirm
our previous lab findings of miR-10b as a prognostic biomarker.
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