Sabato 27 ottobre 2012 - Pacini Editore

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ciation of Medical Oncology (AIOM) and the Italian Society
of Surgical Pathology and Cytopathology (SIAPEC-IAP).
Following the publication of the guidelines, the scientific
societies started an educational program with presentation
and discussion of the guidelines in several national meetings.
AIOM and SIAPEC also organized an external quality assurance
(EQA) program for EGFR testing that revealed that
EGFR mutation analysis is performed with good quality in
the majority of Italian laboratories. The activity of AIOM and
SIAPEC has significantly contributed to improve EGFR testing
and, more generally, molecular pathology in Italy.
references
1 Sharma SV, Bell DW, Settleman J, et al. Epidermal growth factor
receptor mutations in lung cancer. Nat Rev Cancer 2007;7:169-81.
2 Normanno N, De Luca A, Bianco C, et al. Epidermal growth factor
receptor (EGFR) signaling in cancer. Gene 2006;366:2-16.
3 Marchetti A, Normanno N. Recommendations for mutational analysis
of EGFR in lung carcinoma. Pathologica 2010;102:119-26.
ALK: how and when
A. Marchetti
Center of Predictive Molecular Medicine, Center of Excellence on
Aging, University-Foundation, Chieti, Italy
Genetic lesions that drive the proliferation of cancer cells,
known as driver mutations, can make specific tumors sensitive
to therapeutic inhibitors targeting the mutated pathways.
Several target-based therapies are now available for which
treatment optimization is based on tumor testing for specific
mutations and direct therapies against the mutant target. In
patients with non–small-cell lung cancer (NSCLC), inhibitors
of the epidermal growth factor receptor (EGFR), such as
gefitinib or erlotinib, have produced consistent responses in a
subset of cases carrying activating EGFR mutations 1-3 .
More recently, similarly positive outcomes have been reported
for crizotinib in NSCLCs with rearrangement of the
Anaplastic lymphoma kinase (ALK) gene. ALK gene rearrangements
were described for the first time in 2007 as small
inversions on chromosome 2p inducing a fusion of parts of
the echinoderm microtubule-associated protein-like 4 (EML4)
gene with parts of the ALK gene in NSCLC. The resulting fusion
protein (EML4–ALK), with kinase activity, conferred a
strong proliferative stimulus on the cells. 4,5
Activating mutations or translocations of ALK have been
identified in other types of cancer, including anaplastic largecell
lymphoma, inflammatory myofibroblastic tumour, and
paediatric neuroblastoma. 6-8
Multiple distinct EML4-ALK chimeric variants have been
identified, representing breakpoints within various EML4
exons, all of which are transforming in vitro 9,10 .
ALK rearrangement is present in 2-7% of NSCLC cases and it
is associated with distinct clinicopathological features, including
young age of onset, absent or minimal smoking history,
and adenocarcinoma histology 4 10 11-17 .
ALK rearrangements are in general mutually exclusive with
EGFR and KRAS mutations 18 , consistent with the notion
that ALK rearrangement defines a unique molecular subset
of NSCLC.
Preclinical and clinical studies have shown that cancer cells
harbouring EML4–ALK and other ALK abnormalities are
exquisitely sensitive to ALK inhibitors 11 19 .
Crizotinib (PF-02341066; Pfizer) is a selective oral tyrosine
kinase inhibitor of ALK and MET, which inhibits the tyrosine
phosphorylation of ALK 20 21 .
CONGRESSO aNNualE di aNatOmia patOlOGiCa SiapEC – iap • fiRENzE, 25-27 OttOBRE 2012
In a phase I clinical trial the toxicity profile and the efficacy of
crizotinib were evaluated in a cohort of patients with NSCLC and
ALK rearrangement 22 . The study data were recently updated.
These data led to the accelerated approval of crizotinib, by
the Food and Drug Administration (FDA) in the United States
on 26 August 2011, for the treatment of NSCLC patients in a
locally advanced or metastatic state, who are positive for ALK
rearrangement. The use of crizotinib is restricted to patients
whose tumors result positive to ALK alteration from a test
approved by the FDA (currently the Abbott Vysis ALK Break
Apart FISH Probe Kit, Abbott Molecular Inc., IL, USA) 23 .
On 17 August 2011 the European Medicines Agency (EMEA)
in Europe accepted the regulatory submission of crizotinib for
the treatments of patients with advanced stage, ALK-positive,
pretreated NSCLC.
The analysis of molecular changes of ALK is necessary in order
to choose the best therapeutic strategy in selected NSCLC
patients in stages IIIB and IV which, in the presence of gene
rearrangements, may benefit from treatment with ALK inhibitors.
The ALK test is indicated in NSCLC patients with
histotypes of adenocarcinoma, large cell carcinoma, mixed
tumors with adenocarcinoma, or not otherwise specified
(NOS) NSCLC, which present the highest probability of gene
rearrangements. On the basis of current knowledge, the determination
of ALK alterations may be conducted on a surgical
specimen, or biopsy or cytological samples of the primary
tumor and/or of metastases.
In order to guide therapeutic decisions in NSCLC patients the
genetic analysis of ALK runs alongside EGFR gene mutation
research 24 25 . Various technologies have been developed in
order to study this marker.
Fluorescence in-situ hybridization (FISH) is currently the
elective method for the analysis of ALK gene rearrangements.
This method was in fact used in the clinical trials which led
to the approval for treatment with crizotinib. The current diagnostic-therapeutic
protocol sets a cut-off of 15% rearranged
nuclei to consider a case as “positive” and the patient as a
candidate for treatment. The technology is available in commercial
kits developed for diagnostic use, among which is the
diagnostic kit certified by the FDA, Abbott Vysis (ALK Break
Apart FISH Probe Kit, Abbott Molecular, Inc.). Other commercial
kits are available which are not yet FDA-approved
(e.g. ZytoLight®SPEC ALK/EML4 TriCheckProbe, Zyto-
Vision, Bremerhafen, Germany).
The expression of ALK protein could represent a potential
marker indicating gene rearrangement and/or response to
ALK inhibitors. Introducing a user-friendly and cost-friendly
screening method such as immunostaining, into anatomic
pathology laboratories, is highly desirable. To this end, three
monoclonal antibodies have been developed for research purposes
and reported in the literature, clone 5A4 (Leica/Novocastra,
and prediluted Abcam), clone ALK1 (Dako) and clone
D5F3 (Cell Signaling Technology). Results obtained with
these antibodies in comparative studies using the FISH method
are promising, particularly those obtained with clone 5A4
which recognizes a recombinant protein. However, results are
insufficient to draw definitive conclusions at this time.
RT-PCR can be performed on complementary DNA (cDNA)
obtained by messenger RNA (mRNA) synthesis to highlight
directly the process of fusion of ALK with EML4 or other
proteins, using dedicated primers. The technology is extremely
sensitive and highly specific. Nonetheless, RT-PCR has
numerous disadvantages in its application to clinical practice.
Further clinical studies devoted to the identification of the best
technical procedures required are needed.