351 / 366
Conclusion:
IgG4-RD is a recent entity often combined with other
glomerulonephrites and TIN. The definition of molecular markers could
play a crucial role in disease prognosis and predicting outcome.
E-PS-13-006
NGS in molecular pathology labs routine
V. M. Leitão de Sousa*, A. Alarcão, A. F. Ladeirinha, M. Reis Silva, S.
Balseiro, T. Ferreira, M. J. d
’
Aguiar, L. Teixeira, L. Carvalho
*FMUC, IAP-PM, Coimbra, Portugal
Objective:
Next Generation Sequencing has revolutionized the detection of
gene mutations due to its ability to screen multiple mutations in multiple
genes simultaneously without the need to perform several sequential tests,
overtaking the small biopsies barrier, in the continually raising of targeted
therapy biomarkers, as well as cost effectiveness.
Method:
DNA-extraction from formalin-fixed paraffin-embedded tumours
were analyzed for NGS with a panel of 1825 hotspot mutations in 22 genes
(Oncomine Solid Tumour DNA Kit) in Ion PGM. Mutations were detected
using the Variant Caller plugin, the variant list was verified in the IGV and
only mutations reported in the COSMIC database were reported.
Results:
We studied 43 Lung Adenocarcinomas by NGS (Ion PGM); 37
cases had mutations, 24 of which presented more than one mutation. The
driver mutations were: EGFR, KRAS, PIK3CA, ALK, ERBB2, BRAF,
MET and SMAD4, as expected.
Conclusion:
NGS has became more faster and requires less DNA for
tests including more than two or three diferent genes, giving support to
Molecular Pathology Labs as a robust method, prone to have simpler
complements.
E-PS-13-007
Molecular cytogenetics in a case of paediatric rhabdomyosarcoma;
proffering a better prognosis
K. Kuruppu*, A. Thomas, C. Bowker
*John Radcliffe Hospital, Histopathology, Oxford, United Kingdom
Objective:
Rhabdomyosarcoma, a rare malignancy representing up to
50 % of sarcomas in paediatric patients is broadly categorised into two
common morphological sub-types: embryonal and alveolar. Embryonal
rhabdomyosarcoma survival rate is 90 % compared with alveolar (25 %),
necessitating sub-typing. Morphology and immunohistochemistry pro-
files can overlap, therefore molecular cytogenetics is the best way of
providing a definitive diagnosis. We aim to discuss the diagnostic work
up of an orbital rhabdomyosarcoma focusing on the importance of mo-
lecular diagnostics.
Method:
Review of a case including clinical details, histology, immuno-
histochemistry and molecular cytogenetics in combination with current
literature.
Results:
Fluorescent in situ hybridisation analysis showed no FOXO1
translocation typically found in alveolar rhabdomyosarcoma. Gains in
chromosomes 2, 8, 11 and 12 were found in support of embryonal
rhabdomyosarcoma.
Conclusion:
Molecular and cytogenetic tests proved a vital part of the
diagnostic process of this biopsy and led to a favourable tumour sub-type
proffering a better prognosis.
E-PS-13-008
EGFR exon 20 p.T783A from cell-block - case report
V. M. Leitão de Sousa*, A. Alarcão, A. F. Ladeirinha, M. Reis Silva, S.
Balseiro, T. Ferreira, M. João d
’
Aguiar, L. Teixeira, L. Carvalho
*FMUC, IAP-PM, Coimbra, Portugal
Objective:
Somatic activating mutations in the TK domain of the EGFR
confer tumour sensitivity to tyrosine kinase inhibitors (TKIs). Sensitive
molecular testing strategies become the standard to determine targeted
therapies in cancer patients management.
Method:
DNA-extraction from formalin-fixed paraffin-embedded cell-block
of ganglionar metastization with 50 % tumoural cells representation (66-
years-old man with carcinoma - pulmonary/gastric/pancreatic origin) was
analyzed for EGFR mutations by Next Generation Sequencing in Ion PGM
(exons 18/19/20/21). Library preparation followed Oncomine Solid Tumour
DNA Kit procedure. Results analysis was performed in Torrent Server and
Ion Reporter as the Catalogue of Somatic Mutations in Cancer (COSMIC).
R e s u l t s :
E G F R e x o n 2 0 s h o w e d t h e m u t a t i o n
c.2347A>G;p.(Thr783Ala). This mutation confers tumour sensitivity to
tyrosine kinase inhibitors (TKIs); EGFR exons 18, 19, and 21 and KRAS
gene were wild-type.
Conclusion:
This patient selection was crucial for treatment with EGFR
TKIs. In the literature the c.2347A>G;p.(Thr783Ala) mutation was found
in 1 % of EGFR mutations. The importance of sensitive molecular testing
to detect novel EGFR gene mutations sensitive to TKIs, such as p.V765A,
p.T783A, p.V774A, p.S784P, and p.V769A, are becoming standard. Ion
PGM allows sequencing multiple hotspots within the same/ different genes
in routine and this cell-block was representative.
E-PS-13-009
Optimal fixation conditions and DNA extraction methods for
genotyping of FFPE tissue-derived DNA
I. Ganeva*, K. Dinkova, D. Prangova, M. Gulubova
*Medical Faculty, General and Clinical Pathology, Stara Zagora, Bulgaria
Objective:
Tissue biopsies are routinely fixed in formaldehyde and pre-
served in paraffin blocks (formalin-fixed paraffin-embedded-FFPE) as
this classical procedure preserves tissue structures for an accurate histo-
pathological diagnosis. These samples are invaluable genetic resource for
retrospective molecular genetic analysis. However, FFPE tissue samples
are considered problematic starting material for most of the molecular
genetic techniques due to the relatively low quality of extracted
DNA.Therefore, our study is addresing the effects of tissue fixation pro-
cedures and DNA extraction methods of the DNA obtained for down-
stream analysis.
Method:
Surgical specimens of colorectal cancer were fixed in 10 %
buffered or nonbuffered formalin for 1, 12 to 24 hrs, or 48 to 60 hrs at
4oC or at room temperature (RT). DNA extracted from differently fixed
and subsequently paraffin-embedded tissues was used for genotyping via
PCR-RFLP technique. Three commercial DNA extraction kits were
compared.
Results:
Tissues fixed for 12 to 24 hrs in buffered formalin at 4oC tem-
perature produced DNA with the most optimal quality for downstream
analysis. These samples were successfully amplified with intense signal
bands.
Conclusion:
Conclusively, we have observed that in biomedical research
using DNA from FFPE tissues it is important to control a few pre-
treatment steps: optimal pre fixation time, use of 10 % buffered formalin
and use of cold temperature fixation (4oC) and absolutely avoiding of
acidic pH environment.
E-PS-14-001
Where do these guests come from? Diagnostic approach to metastatic
lymph nodes
Y. Dere*, S. Ekmekci, S. Çelik, O. Ilhan Celik, O. Dere, V. Karakus
*Mugla Sitki Kocman University, Dept. of Pathology, Turkey
E-PS-14 Other Topics
Virchows Arch
(
2017
)
471
(
Suppl 1
):
S1
–
S352
S337