|Year : 2017 | Volume
| Issue : 1 | Page : 231-235
Anaplastic lymphoma kinase status in lung cancers: An immunohistochemistry and fluorescence in situ hybridization study from a tertiary cancer center in India
SS Murthy1, SJ Rajappa2, SD Gundimeda1, KM Mallavarapu2, S Ayyagari2, P Yalavarthi2, D Fonseca1, P Paliwal2, HGR Nair1, V Koppula3, KVVN Raju4, ST Rao4
1 Department of Pathology and Laboratory Medicine, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
2 Department of Medical Oncology, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
3 Department of Radiology, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
4 Department of Surgical Oncology, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India
|Date of Web Publication||1-Dec-2017|
Dr. S S Murthy
Department of Surgical Oncology, Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana
Source of Support: None, Conflict of Interest: None
BACKGROUND AND OBJECTIVES: Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) have shown good concordance for the detection of echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) rearrangement. Since studies reporting FISH/IHC concordance, clinicopathological features, and clinical outcomes of ALK-positive patients from India are lacking, this study was undertaken. MATERIALS AND METHODS: This is a retrospective, observational study of patients with adenocarcinoma of the lung on whom ALK test was performed between March 2013 and December 2015. ALK status was assessed in 341 patients by FISH using Vysis ALK Dual Color Break Apart Rearrangement Probe and IHC using ALK D5F3 clone. Clinicopathological features were noted. Patients were managed as per the standard guidelines. Clinical outcomes – response rate (RR) and progression-free survival (PFS) – were measured. RESULTS: ALK rearrangement was positive in 37 patients (10.9%). ALK positivity was observed more commonly in younger patients with no predilection for any gender or any specific histological subtype. ALK by IHC was highly sensitive (100%), compared to FISH with concordance rate of 94.4%. Thirty one of thirty seven (31 of 37) patients received therapy of which 3 patients received palliative chemotherapy and 28 patients received tyrosine kinase inhibitors (crizotinib/ceritinib). Overall RR observed was 77.4%, and median PFS had not been reached at a median follow-up of 12.5 months. INTERPRETATION AND CONCLUSIONS: We report higher frequency of ALK positivity (10.9%) in patients with adenocarcinoma of the lung. ALK by IHC is more sensitive than FISH for ALK detection with high concordance. These patients had good clinical outcome with TKIs targeting ALK fusion protein.
Keywords: Anaplastic lymphoma kinase D5F3, echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase, fluorescence in situ hybridization, immunohistochemistry, lung cancer
|How to cite this article:|
Murthy S S, Rajappa S J, Gundimeda S D, Mallavarapu K M, Ayyagari S, Yalavarthi P, Fonseca D, Paliwal P, Nair H, Koppula V, Raju K, Rao S T. Anaplastic lymphoma kinase status in lung cancers: An immunohistochemistry and fluorescence in situ hybridization study from a tertiary cancer center in India. Indian J Cancer 2017;54:231-5
|How to cite this URL:|
Murthy S S, Rajappa S J, Gundimeda S D, Mallavarapu K M, Ayyagari S, Yalavarthi P, Fonseca D, Paliwal P, Nair H, Koppula V, Raju K, Rao S T. Anaplastic lymphoma kinase status in lung cancers: An immunohistochemistry and fluorescence in situ hybridization study from a tertiary cancer center in India. Indian J Cancer [serial online] 2017 [cited 2019 Oct 18];54:231-5. Available from: http://www.indianjcancer.com/text.asp?2017/54/1/231/219533
| » Introduction|| |
In the era of personalized medicine, tumor histopathology and molecular assessment of oncogene drivers play a significant role in the prognostication and management of lung cancer patients. These oncogenic drivers include epidermal growth factor receptor, echinoderm microtubule-associated protein-like 4, anaplastic lymphoma kinase (EML4-ALK), KRAS, ROS1, MET, and several other markers. All standard guidelines recommend testing for EML4-ALK fusions to guide patient selection for therapy in all patients with advanced-stage adenocarcinoma of the lung regardless of sex, race, smoking history, or other clinical risk factors. The EML4-ALK fusion in nonsmall cell lung cancer (NSCLC) has been established as the best predictor of response to tyrosine kinase inhibitor (TKI) – crizotinib or ceritinib.
Earlier, fluorescence in situ hybridization (FISH) using the break apart probe was considered the gold standard for detection of EML4-ALK rearrangements as it was able to detect all known ALK-rearrangements and was clinically validated in crizotinib clinical trials. However, this method has its own limitations which include signal instability, scoring difficulties, being labor intensive, and not easily available. Immunohistochemistry (IHC) to detect overexpression of ALK protein is another method to detect ALK rearrangement. It is simple, rapid, and inexpensive, and infrastructure is easily available at most laboratories. Reported sensitivity, specificity, and negative predictive value of IHC range from 67% to 100%, 93%–100%, and >98.9%, respectively (in comparison to FISH as the standard method), and several studies have proven high concordance between these two methods.,,
The prevalence of EML4-ALK gene rearrangement by gold standard FISH in unselected population of patient with adenocarcinoma of the lung has been described to range from 2% to 6.7%.,, Published data on ALK status in India are sparse, especially on IHC (ALK D5F3). There are no data on ALK status by IHC from the Indian subcontinent thus far. Moreover, correlation studies between FISH and IHC from this region are lacking.
Here, we present this study from India reporting incidence of EML-ALK positivity in lung cancer patients using both IHC and FISH, analyzing the concordance between these two methods, clinicopathologic features, and clinical outcomes.
| » Materials and Methods|| |
A retrospective analysis of data from March 2013 to December 2015 on EML4-ALK status in lung cancer patients from the archives of the Department of Laboratory Medicine was done. Approval from the Institutional Scientific Review Board and Ethics Committee was obtained. Samples were assessed for appropriate tumor morphology and the presence of viable tumor versus necrosis before processing for molecular workup. The histology was reviewed by two pathologists trained in pulmonary pathology, and all adenocarcinomas were subtyped according to the College of American Pathologists/International Association for the Study of Lung Cancer/American Society of Clinical Oncology guidelines. IHC was performed judiciously in cases of morphologically unclassifiable lesions. Tissue was prioritized for molecular workup over IHC when adequacy of tissue was an issue and the morphological subtype was clear. ALK testing was validated in this center initially by FISH, followed by IHC. All lung cancer cases which tested positive for ALK protein expression by IHC were confirmed by FISH and vice versa.
Fluorescence in situ hybridization
FISH was performed using Vysis LSI ALK Dual Color Break Apart Rearrangement Probe specific to ALK locus at 2p23 according to manufacturer's instructions. The cell was considered to be ALK negative when the cells have one or more fusion signals of overlapping 5' green and 3' orange signals or when cells have a fusion and a 5'green signal. Test was considered positive if 15% or more of the tumor cells had separate 5'(green) and 3' (orange) probe signals with or without fusion or had fusion and isolated 3' orange signals.
Anaplastic lymphoma kinase expression by immunohistochemistry
ALK protein expression using D5F3 clone was assessed on 4 μm thick FFPE sections mounted on positively charged slide on the fully automated BenchMark XT automated slide stainer (Ventana). NSCLC cases with appropriate known ALK-positive and ALK-negative controls were run. All cases were stained with the OptiView DAB IHC Detection Kit and the OptiView Amplification Kit. A case was considered to be positive for ALK expression when a strong, brown, granular cytoplasmic staining was displayed which may be homogeneous or more heterogeneous in staining intensity. Scoring was done by a two-tier system (positive and negative). When the specimen showed the absence of strong granular cytoplasmic staining, the specimen was considered negative for ALK D5F3 expression.
Patients were treated with either upfront TKI (crizotinib, ceritinib) or platinum-based doublet palliative chemotherapy as per the standard guidelines. Response assessment was done as per the RECIST 1.1 criteria. Kaplan–Meier curve was plotted for progression-free survival (PFS) using GraphPad Prism software.
| » Results|| |
Three hundred and sixty-one suspected lung cancer cases were screened for EML4-ALK. Twenty cases (5.5%) were excluded from the study. Biopsy was performed in 251 patients (73.60%) in the hospital while 90 (26.4%) consisted of paraffin blocks from tissues processed elsewhere. The tissue submitted for analysis comprised primary tumor from the lung in 230 (67.4%) patients and metastatic sites in 111 (32.6%) patients. The specimens included 16 (4.69%) lobectomy specimens and 325 (95.30%) guided core needle biopsies.
After exclusion of 20 patients, EML4-ALK status was assessed in 341 patients. In the initial period (March 2013–July 2014), EML4-ALK rearrangement was detected at our institute by FISH in 230 patients. EML4-ALK rearrangement was positive in 16 patients (6.9%) [Figure 1]. The evaluation of ALK D5F3 expression in 111 lung cancers by IHC (after the technique was standardized at our institute in July 2014) indicated expression in 21 (18.91%) patients [Figure 2]. All the 16 positive cases detected by FISH were positive by IHC. However, only 18 of 21 IHC ALK-positive cases were positive by FISH [Table 1].
|Figure 1: Specimen showing fluorescence in situ hybridization anaplastic lymphoma kinase fusions and separated split 5'green and 3'orange signal pattern break apart gene rearrangement|
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|Figure 2: Intense anaplastic lymphoma kinase D5F3 positivity in adenocarcinoma ×100|
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|Table 1: Comparison between immunohistochemistry and fluorescence in situ hybridization|
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One case of ALK D5F3 positivity could not be confirmed by FISH due to inadequate tissue and two patients were negative by FISH. FISH and IHC results showed concordance in 34 out of 36 cases (94.44%). When both techniques were combined, ALK positivity was noted in 37 of 341 patients (10.9%).
FISH signal patterns and positivity rate of all 34 patients (16 FISH and 18 IHC and FISH-positive) are presented in [Table 2]. Four types of signal patterns were observed. Type I was predominant (70.6%), followed by Type II (17.6%), Type III (5.9%), and Type IV (5.9%).
|Table 2: Data on FISH signal pattern from 34 ALK gene rearranged cases positive by FISH|
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Demographic features (median age, gender) and predominant histological subtype for whole cohort, ALK-positive and ALK-negative patients, are presented in [Table 3].
|Table 3: Demographic features and predominant histological subtype for whole cohort (n=341) , ALK positive and ALK negative patients|
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Most common site of metastasis at presentation was lung nodule (M1a), followed by pleural effusion and skeletal metastasis [Table 4].
Although the cytomorphologic classification has been described in excised specimen, we attempted to subclassify lung adenocarcinomas on limited biopsies. The cytomorphology of 223 primary lung adenocarcinomas and 102 cases at metastatic sites indicated that acinar predominant is the most common histologic subtype among whole cohort as well in ALK-positive patients [Table 3] and [Figure 3].
|Figure 3: Adenocarcinoma of the lung (acinar predominant subtype) (H and E, ×400)|
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Treatment and clinical outcomes
Out of 37 ALK-positive patients, 27 were treated with crizotinib (20 upfront, 7 received chemotherapy while waiting for the molecular test results), 1 patient with ceritinib (part of a clinical trial), 3 patients with palliative chemotherapy only, 3 received best supportive care, and 1 underwent surgery. Patients who could not receive ALK TKI due to financial constraints got either palliative chemotherapy or best supportive care only. Median number of chemotherapy cycles given before TKI was 3 (range, 1–4). Of 31 patients - complete response, partial response (PR), stable disease, and progressive disease were seen in 1, 23 (74.2%), 5 (16.1%), and 2 (6.5%), respectively. Median PFS is not reached at a median follow-up of 12.5 months.
Two cases were ALK D5F3 (IHC) positive/FISH negative. One of these patients presented with brain metastasis, received whole brain radiotherapy followed by crizotinib, had a PR, and had not progressed at a follow-up of 3.5 months. The other patient was a 65-year-old male with early-stage lung cancer and underwent surgery (lobectomy) with curative intent and hence did not receive TKI.
| » Discussion|| |
Assessment of EML4-ALK gene rearrangement/ALK protein expression in advanced stages of lung cancer has become standard of care for the management of advanced NSCLC patients. This study is one of the first hospital-based series on patients with EML4-ALK rearrangement in NSCLC from India that has analyzed clinicopathologic and molecular characteristics along with concordance between the two methodologies for detection – FISH and IHC and clinical outcomes. ALK positivity was noted in 37 (10.9%) patients, which is higher than reported by previous Indian studies (2.7%–5.4%) and most international studies (2%–6.7%),, although some studies from Asia have reported that incidence rate ranges from 5.8% to 10%. Although we do not have a valid reason to explain this higher incidence, possible explanation would include ethnic/geographic differences. Zhou et al. cited that IHC can find some ALK-positive cases which would be missed by FISH only.
ALK-positive patients were younger compared to ALK negative (median age - 50 vs. 60 years) and showed no predilection toward any gender (females 51.4% vs. 48.6% males) which is at variance with previous studies. Most studies described increased rate of ALK-positive tumors in women. Cytomorphology on limited biopsies showed predominance of acinar subtype among ALK-positive cases (50%), which was similar to the ALK-negative cohort. However, a specific predilection toward a particular subtype could not be established. Inamura et al. and other studies described combined acinar and cribriform patterns, solid, micropapillary, and papillary-predominant histologic patterns and tumor cells with a signet ring or hepatoid cytomorphology in ALK-positive cohorts, suggesting that no particular histotype is strongly predictive of ALK rearrangement.
Advanced stage at presentation (94.6%), more frequent sites of metastases, and higher incidence of the liver (25.7%) and brain (22%) metastasis at presentation seen in our study were similar to other reported studies.
The present study showed a sensitivity of 100% and concordance rate of 94.44% between IHC and FISH [Table 1], reinforcing the fact that IHC can replace FISH. None of the patients were FISH positive/IHC negative while there were two patients who had IHC positive/FISH negative. One of the two cases, which was ALK IHC positive/FISH negative, showed good response to crizotinib. The other patient had operable lung cancer and hence underwent surgery. One study demonstrated that some FISH-negative but IHC-positive lung cancers did harbor the translocation events as confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Zhou et al. reported three IHC-positive/FISH-negative cases which were ALK positive by RT-PCR.
Borderline cases by FISH have been demonstrated in 8% of NSCLC cases, with the rearrangement-positive cells falling within the range of 10%–20%. Yoshida and Varella-Garcia recommended that recounting of cells, especially in tumor cells using the two-step scoring algorithm and the final interpretation, may be either positive or negative using the 15% cutoff, and confirmation with other modalities IHC or RT-PCR, for further workup. In the present study, 4 of 230 (1.7%) had a borderline signal pattern and counting of additional cells and performing IHC on another block resolved the borderline status.
Preanalytical challenges with reference to FISH include limited biopsies, nature of fixative, quality of wax, prioritization of tissues for morphological/molecular characterization, and training of personnel. Other challenges that impeded ALK testing/enumeration of signals include low cellularity, necrosis and fibrosis in specimen, autofluorescence which affects probe hybridization and signals interpretation. Such issues were encountered in 15 out of 361 (4.1%). The signal patterns seen in ALK-rearranged cases [Table 2] were similar to what has been previously reported.
TKI (crizotinib or ceritinib) is standard of care for patients with ALK-positive advanced lung cancer. In our study, all patients who received TKI (21 upfront, 7 after chemotherapy) had 96.4% overall response which is higher than reported in most of the studies. It may be due to a small number of cases in our study. Median PFS was not reached at a median follow-up of 12.5 months and 1-year progression-free rate is 71% [Figure 4].
Limitations of this study include lack of ALK analysis by both the techniques in ALK-negative cases to study better, concordance between both techniques, and lack of control over preanalytical issues in paraffin blocks processed elsewhere. Studies on a larger scale using IHC to detect ALK are necessary to ascertain the true positivity rate from the subcontinent.
| » Conclusions|| |
Higher ALK positivity (10.9%) and high concordance between IHC and FISH in lung adenocarcinomas were noted. IHC using ALK D5F3 could pick up more cases. Therefore, more patients were eligible for treatment using ALK inhibitors resulting in better outcomes.
We acknowledge the technical assistance provided by Mr. Ravinder, Mrs. Ajitha, Mrs. M. Padma Rani from the Pathology Department.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]