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Prognostic biomarker for NSCLC and Cancer Metastasis

Posted in Cancer and Current Therapeutics, Immunodiagnostics, Lung and pulmonology, tagged 4MiR‐515‐5p, breast cancer, CD24, immunohistochemistry, liquid biopsy, MARK4, Membranous CD24 expression, microRNAs, Microtubule Affinity‐Regulating Kinase, monoclonal antibody SWA11, Non-small cell lung cancer, NSCLC, Prognostic marker on March 24, 2016| Leave a Comment »

Prognostic biomarker for NSCLC and Cancer Metastasis

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Membranous CD24 expression as detected by the monoclonal antibody SWA11 is a prognostic marker in non-small cell lung cancer patients

Michael Majores, Anne Schindler, Angela Fuchs, Johannes Stein, Lukas Heukamp, Peter Altevogt and Glen Kristiansen

BMC Clinical Pathology201515:19   http://dx.doi.org:/10.1186/s12907-015-0019-z

Background    Lung cancer is one of the most common malignant neoplasms worldwide and has a high mortality rate. To enable individualized therapy regimens, a better understanding of the molecular tumor biology has still to be elucidated. The expression of the cell surface protein CD24 has already been claimed to be associated with shorter patient survival in non-small cell lung cancer (NSCLC), however, the prognostic value and applicability of CD24 immunostaining in paraffin embedded tissue specimens has been questioned due to the recent acknowledgement of restricted epitope specificity of the commonly used antibody SN3b.   Methods    A cohort of 137 primary NSCLC cases was immunostained with a novel CD24 antibody (clone SWA11), which specifically recognizes the CD24 protein core and the resulting expression data were compared with expression profiles based on the monoclonal antibody SN3b. Furthermore, expression data were correlated to clinico-pathological parameters. Univariate and multivariate survival analyses were conducted with Kaplan Meier estimates and Cox regression, respectively. Results    CD24 positivity was found in 34 % resp. 21 % (SN3b) of NSCLC with a membranous and/or cytoplasmic staining pattern. Kaplan-Meier analyses revealed that membranous, but not cytoplasmic CD24 expression (clone SWA11) was associated with lympho-nodular spread and shorter overall survival times (both p < 0.05). CD24 expression established by SN3b antibodies did not reveal significant clinicopathological correlations with overall survival, neither for cytoplasmic nor membranous CD24 staining.  Conclusions    Membranous CD24 immunoreactivity, as detected with antibody clone SWA11 may serve as a prognostic factor for lymphonodular spread and poorer overall survival. Furthermore, these results corroborate the importance of a careful distinction between membranous and cytoplasmic localisation, if CD24 is to be considered as a potential prognostic biomarker.

 

Lung cancer is a major cause of carcinoma related death, being responsible for 17.8 % of all cancer deaths and accounting for more than a million deaths worldwide per year [1]. Despite intense studies to improve therapy options, its prognosis has remained poor with a 5-year overall survival rate of less than 15 % [2].

In the past decade, the largest subgroup of lung cancer, i.e. non-small cell lung cancer (NSCLC), has been subjected to exerted research for a better understanding of the underlying molecular biology of lung cancer. More than ten years ago, CD24 has already been suggested as a novel and promising biomarker for carcinoma progression in NSCLC [3] and several groups have confirmed this finding on protein and transcript level [2, 4]. CD24 is a highly glycosylated protein, that binds to the cell surface through a GPI (glycosyl-phosphatidylinositol)-anchor and functions as a cell adhesion molecule and is involved in cell-cell-interaction via its P-selectin binding site [5]. CD24 has been found to be expressed by pre-B-lymphocytes [5]. It is assumed that CD24-positive cells can attach more easily to platelets and activated endothelial cells [6, 7]. Notably, CD24 has also been observed in many human carcinomas, such as ovarian cancer, renal cell cancer, breast cancer and NSCLC [3, 8–12]. In epithelial ovarian cancer high scores of cytoplasmic CD24 were highly predictive of shorter patient survival times (mean 97.8 vs. 36.5 months), whereas membranous CD24 expression seemed to have no influence on survival times. Interestingly, CD24 positivity (membranous or cytoplasmic) of prostate cancer samples was significantly associated to younger patient age and higher pT stages and a higher 3-year prostate-specific antigen (PSA) relapse rate compared with CD24-negative tumours.

In patients with gallbladder carcinoma, tumors with up-regulation of CD24 revealed lymph node metastasis and lymphovascular invasion more frequently. Moreover, up-regulation of CD24 tended to show deeper invasion depth and higher TNM stage [13]. Together, these findings support CD24 as a prognostic marker for carcinoma progression and poorer survival.

Despite these intriguing findings, major concerns regarding a lack of epitope specificity of the commonly used monoclonal antibody SN3b have been raised [14]. Recent findings indicate that the mAb (monoclonal antibody) SN3b does not bind to the protein core itself, but binds to a glycan structure that decorates the CD24 molecule. On the one hand, this motif is not present on all forms of CD24 and—on the other hand—it can be present in other epitopes irrespective of CD24 [14]. These limitations underline the need for more specific CD24 antibodies, such as the mAb SWA11 antibody that has been suggested to be more specific as it binds to the protein core [14].

As CD24 is a promising biomarker for the risk assessment of disease progression, the goal of the present study was to investigate CD24 expression in NSCLC using the novel, more specific monoclonal antibody (mAb) SWA11. Special emphasis was put on the comparison of SN3b- and SWA11-mediated CD24 detection regarding a) the subcellular distribution of CD24 expression (i.e. membranous versus cytoplasmic expression) and b) its correlation with various clinicopathological features including patient survival times.

Table 1

Clinicopathological characteristics of the NSCLC cohort

		AC	SCC
		N (%)	N (%)
Tumour stage (pT)
	1	29 (21.2 %)	5 (3.6)
	2	51 (37.2 %)	23 (16.8 %)
	3	6 (4.4 %)	6 (4.4 %)
	4	1 (0.7 %)	0 (0 %)
Nodal Status (pN)	0	37 (27.0 %)	15 (10.9 %)
	1	15 (10.9 %)	9 (6.6 %)
	2	14 (10.2 %)	3 (2.2 %)
	3	1 (0.7 %)	0 (0.0 %)
Grading (G)	1	5 (3.6 %)	0 (0.0 %)
	2	41 (29.9 %)	16 (11.6 %)
	3	44 (32.1 %)	17 (12.4 %)
Mean age at surgery		64,2	64,56
(median age)		(65)	(67)
Sex (m:w)		68:34	30:5
Median OS (months)		52	24
(SD; 95 % CI [months])		(±23.7; 5.5– 98.5)	(± 12.8;0.0– 49.0)

 

Immunohistochemical detection of CD24 expression using clone SWA11 and SN3b

Using the mAb SWA11, 47 of 137 (34.3 %) NSCLC revealed CD24 expression (either cytoplasmic or membranous) (Table 2). CD24 expression was observed more frequently in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). In AC cytoplasmic expression was observed more frequently than membranous expression. In SCC, both cyptoplasmic and membranous expression was rare. Normal lung parenchyma (i.e. alveolar surface cells) showed no expression of CD24. Bronchial epithelium showed a strong membranous and cytoplasmic staining of the brush border (Fig. 1).

Table 2

Cytoplasmic and membranous expression of CD24

SWA11 (mAb clone)			SN3b (mAB clone)
	AC	SCC		AC	SCC
Cytoplasmic	N (%)	N (%)	Cytoplasmic	N (%)	N (%)
0	45 (32.6 %)	19 (13.8 %)	0	76 (55.1 %)	31 (22.5 %)
1	22 (15.9 %)	8 (5.8 %)	1	12 (8.7 %)	1 (0.7 %)
2	17 (12.3 %)	4 (2.9 %)	2	7 (5.1 %)	2 (1.4 %)
3	18 (13.0 %)	4 (2.9 %)	3	1 (0.7 %)	0 (0 %)
	AC	SCC		AC	SCC
Membranous	N (%)	N (%)	Membranous	N (%)	N (%)
0	68 (49.3 %)	21 (15.2 %)	0	64 (46.4 %)	30 (21.7 %)
1	21 (15.2 %)	5 (3.6 %)	1	10 (7.2 %)	2 (1.4 %)
2	8 (5.8 %)	4 (2.9 %)	2	12 (8.7 %)	2 1.4 %)
3	5 (3.6 %)	5 (3.6 %)	3	10 (7.2 %)	0 (0 %)

Staining intensities are determined as follows:

0: negative or equivocal, 1: weak, 2: moderate and 3: strong CD24 staining

 

Fig 1

The immunohistochemical characterization reveals membranous and/or cytoplasmic CD24 (mAb SWA11) expression. Strong cytoplasmic CD24 expression is found in a proportion of both AC (a) and SCC (b, d) specimens. Membranous CD24 expression can be pronounced with only scant or even absent cytoplasmic staining as shown in the AC (c). Also, both membranous and cytoplasmic CD24 detection can be found in some instances (d), the insert is showing the corresponding squamous carcinoma in-situ with membranous staining. Simultaneous membranous and cytoplasmic CD24 expression is also found in AC specimens (e, f). In normal tissue, alveolar epithelial cells do not express CD24 (g), whereas CD24 staining is found at the apical cell membrane of bronchial respiratory epithelia (h)

Using the mAb SN3b, 29 of 137 (21.2 %) NSCLC revealed CD24 expression (either cytoplasmic or membranous) (Table 2). As above, CD24 expression was observed more frequently in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). However, in contrast to mAb SWA11 cytoplasmic expression was observed less frequently than membranous expression in AC. In SCC, both cytoplasmic and membranous expression was rare. Normal lung parenchyma (i.e. alveolar surface cells) showed a distinct membranous immunoreactivity. Bronchial epithelium revealed both membranous and cytoplasmic staining of CD24.

Correlation between SWA11 and SN3b: As SWA11 and SN3b detect different epitopes, we evaluated the correlation of the immunohistochemical staining patterns. Of 132 NSCLC specimens with matched expression data, only 9 specimens (6.8 %) revealed a concordant CD24 expression. Of these cases, 4 cases revealed a concordant cytoplasmic staining and another 5 cases revealed a concordant membranous CD24 expression. Statistically, no significant correlation between the two mAb could be observed (cc = −0.63, p = 0.470; Fisher’s exact test p = 0.665). The correlation of cytoplasmic and membranous expression (for each antibody) was as follows: cc = 0.475 (p < 0.05) for SWA11 (n = 108) and cc = 0.140 (p = 0.11) for SN3b (n = 103).

Survival analyses

Recent studies indicate that CD24 expression is associated with tumor progression and poorer survival rates. Therefore, we performed follow up analyses with a special emphasis on 1) the prognostic value of mAb SWA11 in dependence on subcellular staining characteristics and 2) the prognostic values of different clinicopathological parameters:

Prognostic value of CD24 in Kaplan Meier Analyses

Only membranous CD24 (SWA11) staining revealed significantly poorer survival rates (median overall survival 21 vs. 52 months; p = 0.005) as illustrated in Fig. 2. In contrast, cytoplasmic CD24 (SWA11) staining did not affect the survival rates (median OS 34 vs. 35 months; p = 0.884) (Table 3). When stratifying the cohort into SCC (n = 35) and AC (n = 102) in Kaplan Meier analyses, membranous CD24 (SWA11) expression did not affect patients’ survival, neither in SCC (p = 0.243) nor AC (p = 0.135) (Table 3), probably due to the small number of observations (Fisher exact test: p > 0.05). After stratification for AC subtypes, membranous CD24 expression (SWA11) showed a tendency towards an association with poorer survival in acinar subtype AC, but failed significance (p = 0.328).

Fig 2

Survival analysis. Kaplan-Meier curves according to SWA11 expression. Cases with moderate to strong expression were bundled in a ‘high expression’ and cases with negative or weak expression in a ‘low expression’ group. Membranous expression of CD24 detected by SWA11 proved to be an independent marker for shorter survival times in NSCLC (p = 0.005)

Table 3

Univariate survival analysis

SWA11	No. of cases	Mean survival time	Median survival time	p-value
		(months +/− s.e.)	(months +/− s.e.)
Mem CD24
Negative	76	84.833 +/− 10.395	52.000 +/− 27.030	0.005
Positive	16	27.925 +/− 6.379	21.000 +/− 4.000
Cyto CD24
Negative	66	75.209 +/− 10.577	35.000 +/− 12.422	0.884
Positive	26	60.540 +/− 11.551	34.000 +/− 12.196
Total CD24
Negative	64	76.972 +/− 10.841	35.000 +/− 13.726	0.633
Positive	28	57.535 +/− 10.895	34.000 +/− 9.303
SCC
Mem CD24 negative	16	52.063 +/− 14.668	16.000 +/− 16.000	0.243
Mem CD24 positive	7	21.571 +/− 7.201	24.000 +/− 23.568
AC
Mem CD24 negative	59	88.953 +/− 11.631	56.000 +/− 22.885	0.135
Mem CD24 positive	8	39.167 +/− 11.674	21.000 +/− 8.485
pN0	31	103.641 +/− 14.940	93.000 +/− 28.224	0.012
pN1+	30	54.911 +/− 10.646	26.000 +/− 0.983

 

…..

Univariate survival analysis according to the Cox regression model (mAb SWA11)

	Beta	HR (hazard ratio)	95 % CI of HR	P-value
SWA11 mem all	0.856	2.353	1.268–4.364	0.007
pN	0.963	2.620	1.389–4.943	0.003
pT	0.844	2.325	1.279–4.224	0.006
Tumour type	0.975	2.651	1.999–3.517	0.000

Table 5

Multivariate survival analysis according to the Cox regression model (mAb SWA11)

	Beta	HR (hazard ratio)	95 % CI of HR	P-value
SWA11 mem all	0.944	2.571	1.211–5.458	0.014
pN	0.737	2.091	1.087–4.021	0.027
pT	0.587	1.799	0.755–4.283	0.185

 

…..

In the present study, we have analyzed immunohistochemical staining characteristics and the prognostic value of CD24 expression in NSCLC with a special emphasis on the comparison of the CD24 antibodies SWA11 and SN3b. The most important result of our study is that the prognostic relevance of CD24 is critically dependent on the careful consideration of sub-cellular compartments and the epitope specificity of the antibody used.

Overall, about one third of the NSCLC cohort revealed a significant CD24 expression (either cytoplasmic or membranous). These results are in line with the findings of other studies. In another NSCLC cohort, CD24 (SN3b) expression was found in 33 % of the samples (87 of 267 cases) [2]. Consistent with those results, we have found similar rates of high CD24 expression levels (35 % of the cases) for SWA11. Originally, we would have expected lower rates than those found by Lee et al, as they used the antibody SN3b, that also recognizes yet unidentified other glycoproteins next to CD24. Furthermore, they used whole mount sections instead of tissue microarrays. A possible explanation for rather equal detection rates would be the fact that it has been demonstrated that the epitope recognized by SN3b is indeed present in CD24, but is not found in all glycoforms of CD24 [14]. In contrast to the commonly used mAb SN3b, mAb SWA11 binds to the protein core of CD24 and does not depict other glycan moieties next to CD24. The protein core of CD24 is linear, consisting of the amino acid sequence leucine-proline-alanine (LAP) next to a glycosyl-phosphatidylinositol anchor [15].

CD24 expression has been associated with disease progression and cancer-related death in the majority of malignant tumors [2, 3, 16, 17], although a caveat to these data is that most of these studies are based on the supposedly less specific CD24 clone SN3b. Lee et al demonstrated a significant association between CD24-high expression (SN3b) and shorter patient survival times. Furthermore, Lee and colleagues and ourselves in former studies referred the results to cytoplasmic CD24 expression [2, 3].

Switching Off Cancers’ Ability to Spread

http://www.technologynetworks.com/rnai/news.aspx?ID=189704

A key molecule in breast and lung cancer cells can help switch off the cancers’ ability to spread around the body.

The findings by researchers at Imperial College London, published in the journal EMBO Reports, may help scientists develop treatments that prevent cancer travelling around the body – or produce some kind of test that allows doctors to gauge how likely a cancer is to spread. During tumour growth, cancer cells can break off and travel in the bloodstream or lymph system to other parts of the body, in a process called metastasis.

Patients whose cancers spread tend to have a worse prognosis, explains Professor Justin Stebbing, senior author of the study from the Department of Surgery and Cancer at Imperial: “The ability of a cancer to spread around the body has a large impact on a patient’s survival. However, at the moment we are still in the dark about why some cancers spread around the body – while others stay in one place. This study has given important insights into this process.”

The researchers were looking at breast and lung cancer cells and they found that a protein called MARK4 enables the cells to break free and move around to other parts of the body, such as the brain and liver. Although scientist are still unsure how it does this, one theory is it affects the cell’s internal scaffolding, enabling it to move more easily around the body. The team found that a molecule called miR-515-5p helps to silence, or switch off, the gene that produces MARK4.

In the study, the team used human breast cancer and lung cancer cells to show that the miR-515-5p molecule silences the gene MARK4. They then confirmed this in mouse models, which showed that increasing the amount of miR-515-5p prevents the spread of cancer cells. The findings also revealed that the silencer molecule was found in lower levels in human tumours that had spread around the body. The team then also established that patients with breast and lung cancers whose tumours had low amounts of these silencer molecules – or high amounts of MARK4 – had lower survival rates.

Researchers are now investigating whether either the MARK4 gene or the silencer molecule could be targeted with drugs. They are also investigating whether these molecules could be used to develop a test to indicate whether a patient’s cancer is likely to spread. Professor Stebbing said: “In our work we have shown that this silencer molecule is important in the spread of cancer. This is very early stage research, so we now need more studies to find out more about this molecule, and if it is present in other types of cancer.”

Dr Olivier Pardo, lead author of the paper, also from the Department of Surgery and Cancer at Imperial, added: “Our work also identified that MARK4 enables breast and lung cancer cells to both divide and invade other parts of the body. These findings could have profound implications for treating breast and lung cancers, two of the biggest cancer killers worldwide.” The study was supported by the NIHR Imperial Biomedical Research Centre, the Medical Research Council, Action Against Cancer and the Cancer Treatment and Research Trust.

 

‘Silencer molecules’ switch off cancer’s ability to spread around body

by Kate Wighton

Scientists have revealed that a key molecule in breast and lung cancer cells can help switch off the cancers’ ability to spread around the body

The findings by researchers at Imperial College London, published in the journal EMBO Reports, may help scientists develop treatments that prevent cancer travelling around the body – or produce some kind of test that allows doctors to gauge how likely a cancer is to spread.

During tumour growth, cancer cells can break off and travel in the bloodstream or lymph system to other parts of the body, in a process called metastasis.

Patients whose cancers spread tend to have a worse prognosis, explains Professor Justin Stebbing, senior author of the study from the Department of Surgery and Cancer at Imperial: “The ability of a cancer to spread around the body has a large impact on a patient’s survival. However, at the moment we are still in the dark about why some cancers spread around the body – while others stay in one place. This study has given important insights into this process.”

The researchers were looking at breast and lung cancer cells and they found that a protein called MARK4 enables the cells to break free and move around to other parts of the body, such as the brain and liver. Although scientist are still unsure how it does this, one theory is it affects the cell’s internal scaffolding, enabling it to move more easily around the body.

 

miR‐515‐5p controls cancer cell migration through MARK4 regulation

Olivier E Pardo, Leandro Castellano, Catriona E Munro, Yili Hu, Francesco Mauri,Jonathan Krell, Romain Lara, Filipa G Pinho, Thameenah Choudhury, Adam EFrampton, Loredana Pellegrino, Dmitry Pshezhetskiy, Yulan Wang, JonathanWaxman, Michael J Seckl, Justin Stebbing    

EMBO reports (2016) embr.201540970   http://embor.embopress.org/content/early/2016/02/10/embr.201540970     http://dx.doi.org:/10.15252/embr.201540970| Published online 10.02.2016

Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients’ survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers.

miR‐515‐5p inhibits cancer progression, cell migration and metastasis through its direct target MARK4, a regulator of the cytoskeleton and cell motility. Moreover, reduced miR‐515‐5p and increased MARK4 levels in metastatic lung and breast cancer correlate with poor patient prognosis.

• MARK4 down‐regulation promotes microtubule polymerisation.

• Increased cell spreading downstream of miR‐515‐5p overexpression or MARK4 silencing hinders cell motility and invasiveness.

• miR‐515‐5p overexpression or MARK4 silencing prevent organ colonisation by circulating tumour cells.

• MARK4 inhibitors may represent novel therapeutic agents to control cancer dissemination.breasat cancer

 

Liquid Biopsy for NSCLC

http://www.technologynetworks.com/Diagnostics/news.aspx?ID=190276

‘Liquid biopsy’ blood test accurately detects key genetic mutations in most common form of lung cancer, study finds.

A simple blood test can rapidly and accurately detect mutations in two key genes in non-small cell lung tumors, researchers at Dana-Farber Cancer Institute and other institutions report in a new study – demonstrating the test’s potential as a clinical tool for identifying patients who can benefit from drugs targeting those mutations. The test, known as a liquid biopsy, proved so reliable in the study that Dana-Farber/Brigham and Women’s Cancer Center (DF/BWCC) expects to offer it soon to all patients with non-small cell lung cancer (NSCLC), either at the time of first diagnosis or of relapse following previous treatment. NSCLC is the most common form of lung cancer, diagnosed in more than 200,000 people in the United States each year, according to the American Cancer Society. An estimated 30 percent of NSCLC patients have mutations in either of the genes included in the study, and can often be treated with targeted therapies. The study is being published online today by the journal JAMA Oncology. The liquid biopsy tested in the study – technically known as rapid plasma genotyping – involves taking a test tube-full of blood, which contains free-floating DNA from cancer cells, and analyzing that DNA for mutations or other abnormalities. (When tumor cells die, their DNA spills into the bloodstream, where it’s known as cell-free DNA.) The technique, which provides a “snapshot” of key genetic irregularities in a tumor, is a common tool in research for probing the molecular make-up of different kinds of cancers. “We see plasma genotyping as having enormous potential as a clinical test, or assay – a rapid, noninvasive way of screening a cancer for common genetic fingerprints, while avoiding the challenges of traditional invasive biopsies,” said the senior author of the study, Geoffrey Oxnard, MD, thoracic oncologist and lung cancer researcher at Dana-Farber and Brigham and Women’s Hospital. “Our study was the first to demonstrate prospectively that a liquid biopsy technique can be a practical tool for making treatment decisions in cancer patients. The trial was such a success that we are transitioning the assay into a clinical test for lung cancer patients at DF/BWCC.” The study involved 180 patients with NSCLC, 120 of whom were newly diagnosed, and 60 of whom had become resistant to a previous treatment, allowing the disease to recur. Participants’ cell-free DNA was tested for mutations in the EGFR and KRAS genes, and for a separate mutation in EGFR that allows tumor cells to become resistant to front-line targeted drugs. The test was performed with a technique known as droplet digital polymerase chain reaction (ddPCR), which counts the individual letters of the genetic code in cell-free DNA to determine if specific mutations are present. Each participant also underwent a conventional tissue biopsy to test for the same mutations. The results of the liquid biopsies were then compared to those of the tissue biopsies. The data showed that liquid biopsies returned results much more quickly. The median turnaround time for liquid biopsies was three days, compared to 12 days for tissue biopsies in newly diagnosed patients and 27 days in drug-resistant patients. Liquid biopsy was also found to be highly accurate. In newly diagnosed patients, the “predictive value” of plasma ddPCR was 100 percent for the primary EGFR mutation and the KRAS mutation – meaning that a patient who tested positive for either mutation was certain to have that mutation in his or her tumor. For patients with the EGFR resistance mutation, the predictive value of the ddPCR test was 79 percent, suggesting the blood test was able to find additional cases with the mutation that were missed using standard biopsies. “In some patients with the EGFR resistance mutation, ddPCR detected mutations missed by standard tissue biopsy,” Oxnard remarked. “A resistant tumor is inherently made up of multiple subsets of cells, some of which carry different patterns of genetic mutations. A single biopsy is only analyzing a single part of the tumor, and may miss a mutation present elsewhere in the body. A liquid biopsy, in contrast, may better reflect the distribution of mutations in the tumor as a whole.” When ddPCR failed to detect these mutations, the cause was less clear-cut, Oxnard says. It could indicate that the tumor cells don’t carry the mutations or, alternatively, that the tumor isn’t shedding its DNA into the bloodstream. This discrepancy between the test results and the presence of mutations was less common in patients whose cancer had metastasized to multiple sites in the body, researchers found. The ddPCR-based test, or assay, was piloted and optimized for patients at the Translational Resarch lab of the Belfer Center for Applied Cancer Science at Dana-Farber. It was then validated for clinical use at Dana-Farber’s Lowe Center for Thoracic Oncology. An advantage of this form of liquid biopsy is that it can help doctors quickly determine whether a patient is responding to therapy. Fifty participants in the study had repeat testing done after starting treatment for their cancer. “Those whose blood tests showed a disappearance of the mutations within two weeks were more likely to stay on the treatment than patients who didn’t see such a reduction,” said the study’s lead author, Adrian Sacher, MD, of Dana-Farber and Brigham and Women’s Hospital. And because tumors are constantly evolving and acquiring additional mutations, repeated liquid biopsies can provide early detection of a new mutation – such as the EGFR resistance mutation – that can potentially be treated with targeted agents. “The study data are compelling,” said DF/BWCC pathologist Lynette Sholl, MD, explaining the center’s decision to begin offering ddPCR-based liquid biopsy to all lung cancer patients. “We validated the authors’ findings by cross-comparing results from liquid and tissue biopsies in 34 NSCLC patients. To work as a real-world clinical test, liquid biopsy needs to provide reliable, accurate data and be logistically practical. That’s what we’ve seen with the ddPCR-based blood test. “The test has great utility both for patients newly diagnosed with NSCLC and for those with a recurrence of the disease,” she continued. “It’s fast, it’s quantitative (it indicates the amount of mutant DNA in a sample), and it can be readily employed at a cancer treatment center.” The co-authors of the study are Cloud Paweletz, PhD, Allison O’Connell, BSc, and Nora Feeney, BSc, of the Belfer Center for Applied Cancer Science at Dana-Farber; Ryan S. Alden BSc, and Stacy L. Mach BA, of Dana-Farber; Suzanne E. Dahlberg, PhD, of Dana-Farber and Harvard T.H. Chan School of Public Health; and Pasi A. Jänne, MD, PhD, of Dana-Farber, the Belfer Center, and Brigham and Women’s Hospital.

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Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Posted in Biological Networks, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Prevention: Research & Programs, Computational Biology/Systems and Bioinformatics, Gene Regulation and Evolution, Genome Biology, Health Economics and Outcomes Research, Personalized and Precision Medicine & Genomic Research, tagged antitumor therapy, Bioinformatics, Cancer - General, Cell Biology, Computational Biology/Systems and Bioinformatics, DNA, DNA Sequencing, driver mutations, gene expression, genetics, genome, genomics, informatics, Lung cancer, mutation, mutational analysis, National Institutes of Health, Non-small cell lung cancer, Personalized medicine, Proceedings of the National Academy of Sciences of the United States of America, Prostate cancer, research, smoking, The Cancer Genome Atlas (TCGA), The Clinical Lung Cancer Genome Project (CLCGP), whole exome sequencing on September 5, 2014| Leave a Comment »

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Curator, Writer: Stephen J. Williams, Ph.D.

UPDATED 10/10/2021

(photo credit: cancer.gov)

A report Lung Cancer Genome Surveys Find Many Potential Drug Targets, in the NCI Bulletin,

http://www.cancer.gov/ncicancerbulletin/091812/page2

summarizes the clinical importance of five new lung cancer genome sequencing projects. These studies have identified genetic and epigenetic alterations in hundreds of lung tumors, of which some alterations could be taken advantage of using currently approved medications.

The reports, all published this month, included genomic information on more than 400 lung tumors. In addition to confirming genetic alterations previously tied to lung cancer, the studies identified other changes that may play a role in the disease.

Collectively, the studies covered the main forms of the disease—lung adenocarcinomas, squamous cell cancers of the lung, and small cell lung cancers.

“All of these studies say that lung cancers are genomically complex and genomically diverse,” said Dr. Matthew Meyerson of Harvard Medical School and the Dana-Farber Cancer Institute, who co-led several of the studies, including a large-scale analysis of squamous cell lung cancer by The Cancer Genome Atlas (TCGA) Research Network.

Some genes, Dr. Meyerson noted, were inactivated through different mechanisms in different tumors. He cautioned that little is known about alterations in DNA sequences that do not encode genes, which is most of the human genome.

Four of the papers are summarized below, with the first described in detail, as the Nature paper used a multi-‘omics strategy to evaluate expression, mutation, and signaling pathway activation in a large cohort of lung tumors. A literature informatics analysis is given for one of the papers.  Please note that links on GENE names usually refer to the GeneCard entry.

Paper 1. Comprehensive genomic characterization of squamous cell lung cancers[1]

The Cancer Genome Atlas Research Network Project just reported, in the journal Nature, the results of their comprehensive profiling of 230 resected lung adenocarcinomas. The multi-center teams employed analyses of

• microRNA
• Whole Exome Sequencing including
  • Exome mutation analysis
  • Gene copy number
  • Splicing alteration
• Methylation
• Proteomic analysis

Summary:

Some very interesting overall findings came out of this analysis including:

• High rates of somatic mutations including activating mutations in common oncogenes
• Newly described loss of function MGA mutations
• Sex differences in EGFR and RBM10 mutations
• driver roles for NF1, MET, ERBB2 and RITI identified in certain tumors
• differential mutational pattern based on smoking history
• splicing alterations driven by somatic genomic changes
• MAPK and PI3K pathway activation identified by proteomics not explained by mutational analysis = UNEXPLAINED MECHANISM of PATHWAY ACTIVATION

however, given the plethora of data, and in light of a similar study results recently released, there appears to be a great need for additional mining of this CGAP dataset. Therefore I attempted to curate some of the findings along with some other recent news relevant to the surprising findings with relation to biomarker analysis.

Makeup of tumor samples

230 lung adenocarcinomas specimens were categorized by:

Subtype

33% acinar

25% solid

14% micro-papillary

9% papillary

8% unclassified

5% lepidic

4% invasive mucinous
Gender

Smoking status

81% of patients reported past of present smoking

The authors note that TCGA samples were combined with previous data for analysis purpose.

A detailed description of Methodology and the location of deposited data are given at the following addresses:

Publication TCGA Web Page: https://tcga-data.nci.nih.gov/docs/publications/luad_2014/

Sequence files: https://cghub.ucsc.edu

Results:

Gender and Smoking Habits Show different mutational patterns

 

WES mutational analysis

1. a) smoking status

– there was a strong correlations of cytosine to adenine nucleotide transversions with past or present smoking. In fact smoking history separated into transversion high (past and previous smokers) and transversion low (never smokers) groups, corroborating previous results.

→ mutations in groups              Transversion High                   Transversion Low

TP53, KRAS, STK11,                 EGFR, RB1, PI3CA

     KEAP1, SMARCA4 RBM10

 

1. b) Gender

Although gender differences in mutational profiles have been reported, the study found minimal number of significantly mutated genes correlated with gender. Notably:

• EGFR mutations enriched in female cohort
• RBM10 loss of function mutations enriched in male cohort

Although the study did not analyze the gender differences with smoking patterns, it was noted that RBM10 mutations among males were more prevalent in the transversion high group.

Whole exome Sequencing and copy number analysis reveal Unique, Candidate Driver Genes

Whole exome sequencing revealed that 62% of tumors contained mutations (either point or indel) in known cancer driver genes such as:

KRAS, EGFR, BRMF, ERBB2

However, authors looked at the WES data from the oncogene-negative tumors and found unique mutations not seen in the tumors containing canonical oncogenic mutations.

Unique potential driver mutations were found in

TP53, KEAP1, NF1, and RIT1

The genomics and expression data were backed up by a proteomics analysis of three pathways:

1. MAPK pathway
2. mTOR
3. PI3K pathway

…. showing significant activation of all three pathways HOWEVER the analysis suggested that activation of signaling pathways COULD NOT be deduced from DNA sequencing alone. Phospho-proteomic analysis was required to determine the full extent of pathway modification.

For example, many tumors lacked an obvious mutation which could explain mTOR or MAPK activation.

 

Altered cell signaling pathways included:

• Increased MAPK signaling due to activating KRAS
• Higher mTOR due to inactivating STK11 leading to increased proliferation, translation

Pathway analysis of mutations revealed alterations in multiple cellular pathways including:

• Reduced oxidative stress response
• Nucleosome remodeling
• RNA splicing
• Cell cycle progression
• Histone methylation

Summary:

Authors noted some interesting conclusions including:

1. MET and ERBB2 amplification and mutations in NF1 and RIT1 may be unique driver events in lung adenocarcinoma
2. Possible new drug development could be targeted to the RTK/RAS/RAF pathway
3. MYC pathway as another important target
4. Cluster analysis using multimodal omics approach identifies tumors based on single-gene driver events while other tumor have multiple driver mutational events (TUMOR HETEROGENEITY)

Paper 2. A Genomics-Based Classification of Human Lung Tumors[2]

The paper can be found at

http://stm.sciencemag.org/content/5/209/209ra153

by The Clinical Lung Cancer Genome Project (CLCGP) and Network Genomic Medicine (NGM),*,†

Paper Summary

This sequencing project revealed discrepancies between histologic and genomic classification of lung tumors.

Methodology

– mutational analysis by whole exome sequencing of 1255 lung tumors of histologically

defined subtypes

– immunohistochemistry performed to verify reclassification of subtypes based on sequencing data

Results

• 55% of all cases had at least one oncogenic alteration amenable to current personalized treatment approaches
• Marked differences existed between cluster analysis within and between preclassified histo-subtypes
• Reassignment based on genomic data eliminated large cell carcinomas
• Prospective classification of 5145 lung cancers allowed for genomic classification in 75% of patients
• Identification of EGFR and ALK mutations led to improved outcomes

Conclusions:

It is feasible to successfully classify and diagnose lung tumors based on whole exome sequencing data.

Paper 3. Genomic Landscape of Non-Small Cell Lung Cancer in Smokers and Never-Smokers[3]

A link to the paper can be found here with Graphic Summary: http://www.cell.com/cell/abstract/S0092-8674%2812%2901022-7?cc=y?cc=y

Methodology

• Whole genome sequencing and transcriptome sequencing of cancerous and adjacent normal tissues from 17 patients with NSCLC
• Integrated RNASeq with WES for analysis of
  • Variant analysis
  • Clonality by variant allele frequency anlaysis
  • Fusion genes
• Bioinformatic analysis
  • PathScan, KEGG for pathway analysis
  • COSMIC for reported mutations
  • ChimeraScan, defuse, BreakFusion for fusion protein analysis

Results

• 3,726 point mutations and more than 90 indels in the coding sequence
• Smokers with lung cancer show 10× the number of point mutations than never-smokers
• Novel lung cancer genes, including DACH1, CFTR, RELN, ABCB5, and HGF were identified

• Tumor samples from males showed high frequency of MYCBP2 MYCBP2 involved in transcriptional regulation of MYC.

• Variant allele frequency analysis revealed 10/17 tumors were at least biclonal while 7/17 tumors were monoclonal revealing majority of tumors displayed tumor heterogeneity
• Novel pathway alterations in lung cancer include cell-cycle and JAK-STAT pathways
• 14 fusion proteins found, including ROS1-ALK fusion. ROS1-ALK fusions have been frequently found in lung cancer and is indicative of poor prognosis[4].
• Novel metabolic enzyme fusions
• Alterations were identified in 54 genes for which targeted drugs are available.           Drug-gable mutant targets include: AURKC, BRAF, HGF, EGFR, ERBB4, FGFR1, MET, JAK2, JAK3, HDAC2, HDAC6, HDAC9, BIRC6, ITGB1, ITGB3, MMP2, PRKCB, PIK3CG, TERT, KRAS, MMP14

Table. Validated Gene-Fusions Obtained from Ref-Seq Data

Note: Gene columns contain links for GeneCard while Gene function links are to the    gene’s GO (Gene Ontology) function.

GeneA (5′)	GeneB (3′)	GeneA function (link to Gene Ontology)	GeneB function (link to Gene Ontology)		known function (refs)
GRIP1	TNIP1	glutamate receptor IP	transcriptional repressor
SGMS1	STK10	sphingolipid synthesis	ser/thr kinase
RASSF3	TTYH2	GTP-binding protein	chloride anion channel
KDELR2	ROS1, GOPC	ER retention seq. binding	proto-oncogenic tyr kinase
ACSL4	DCAF6	fatty acid synthesis	?
MARCH8	PRKG1	ubiquitin ligase	cGMP dependent protein kinase
APAF1	UNC13B, TLN1	caspase activation	cytoskeletal
EML4	ALK	microtubule protein	tyrosine kinase		♦
EDR3,PHC3	LOC441601	polycomb pr/DNA binding	?
DKFZp761L1918,RHPN2	ANKRD27	Rhophilin (GTP binding pr	ankyrin like
VANGL1	HAO2	tetraspanin family	oxidase
CACNA2D3	FLNB	VOC Ca++ channel	filamin (actin binding)

† Author’s Note:

There has been a recent literature on the importance of the EML4-ALK fusion protein in lung cancer. EML4-ALK positive lung tumors were found to be les chemo sensitive to cytotoxic therapy[5] and these tumor cells may exhibit an epitope rendering these tumors amenable to immunotherapy[6]. In addition, inhibition of the PI3K pathway has sensitized EMl4-ALK fusion positive tumors to ALK-targeted therapy[7]. EML4-ALK fusion positive tumors show dependence on the HSP90 chaperone, suggesting this cohort of patients might benefit from the new HSP90 inhibitors recently being developed[8].

Table. Significantly mutated genes (point mutations, insertions/deletions) with associated function.

Gene	Function
TP53	tumor suppressor
KRAS	oncogene
ZFHX4	zinc finger DNA binding
DACH1	transcription factor
EGFR	epidermal growth factor receptor
EPHA3	receptor tyrosine kinase
ENSG00000205044
RELN	cell matrix protein
ABCB5	ABC Drug Transporter

Table. Literature Analysis of pathways containing significantly altered genes in NSCLC reveal putative targets and risk factors, linkage between other tumor types, and research areas for further investigation.

Note: Significantly mutated genes, obtained from WES, were subjected to pathway analysis (KEGG Pathway Analysis) in order to see which pathways contained signicantly altered gene networks. This pathway term was then used for PubMed literature search together with terms “lung cancer”, “gene”, and “NOT review” to determine frequency of literature coverage for each pathway in lung cancer. Links are to the PubMEd search results.

KEGG pathway Name	# of PUBMed entries containing Pathway Name, Gene ANDLung Cancer
Cell cycle	1237
Cell adhesion molecules (CAMs)	372
Glioma	294
Melanoma	219
Colorectal cancer	207
Calcium signaling pathway	175
Prostate cancer	166
MAPK signaling pathway	162
Pancreatic cancer	88
Bladder cancer	74
Renal cell carcinoma	68
Focal adhesion	63
Regulation of actin cytoskeleton	34
Thyroid cancer	32
Salivary secretion	19
Jak-STAT signaling pathway	16
Natural killer cell mediated cytotoxicity	11
Gap junction	11
Endometrial cancer	11
Long-term depression	9
Axon guidance	8
Cytokine-cytokine receptor interaction	8
Chronic myeloid leukemia	7
ErbB signaling pathway	7
Arginine and proline metabolism	6
Maturity onset diabetes of the young	6
Neuroactive ligand-receptor interaction	4
Aldosterone-regulated sodium reabsorption	2
Systemic lupus erythematosus	2
Olfactory transduction	1
Huntington’s disease	1
Chemokine signaling pathway	1
Cardiac muscle contraction	1
Amyotrophic lateral sclerosis (ALS)	1

A few interesting genetic risk factors and possible additional targets for NSCLC were deduced from analysis of the above table of literature including HIF1-α, mIR-31, UBQLN1, ACE, mIR-193a, SRSF1. In addition, glioma, melanoma, colorectal, and prostate and lung cancer share many validated mutations, and possibly similar tumor driver mutations.

 please click on graph for larger view

Paper 4. Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing[9]

For full paper and graphical summary please follow the link: http://www.cell.com/cell/abstract/S0092-8674%2812%2901061-6

Highlights

• Exome and genome characterization of somatic alterations in 183 lung adenocarcinomas
• 12 somatic mutations/megabase
• U2AF1, RBM10, and ARID1A are among newly identified recurrently mutated genes
• Structural variants include activating in-frame fusion of EGFR
• Epigenetic and RNA deregulation proposed as a potential lung adenocarcinoma hallmark

Summary

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Paper 5. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer[10]

Highlights

• Whole exome and transcriptome (RNASeq) sequencing 29 small-cell lung carcinomas
• High mutation rate 7.4 protein-changing mutations/million base pairs
• Inactivating mutations in TP53 and RB1
• Functional mutations in CREBBP, EP300, MLL, PTEN, SLIT2, EPHA7, FGFR1 (determined by literature and database mining)
• The mutational spectrum seen in human data also present in a Tp53-/- Rb1-/- mouse lung tumor model

 

Curator Graphical Summary of Interesting Findings From the Above Studies

The above figure (please click on figure) represents themes and findings resulting from the aforementioned studies including

questions which will be addressed in Future Posts on this site.

UPDATED 10/10/2021

The following article uses RNASeq to screen lung adenocarcinomas for fusion proteins in patients with either low or high tumor mutational burden. Findings included presence of MET fusion proteins in addition to other fusion proteins irrespective if tumors were driver negative by DNASeq screening.

High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden

Source:

High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden

Ryma Benayed, Michael Offin, Kerry Mullaney, Purvil Sukhadia, Kelly Rios, Patrice Desmeules, Ryan Ptashkin, Helen Won, Jason Chang, Darragh Halpenny, Alison M. Schram, Charles M. Rudin, David M. Hyman, Maria E. Arcila, Michael F. Berger, Ahmet Zehir, Mark G. Kris, Alexander Drilon and Marc Ladanyi

Clin Cancer Res August 1 2019 (25) (15) 4712-4722; DOI: 10.1158/1078-0432.CCR-19-0225

Abstract

Purpose: Targeted next-generation sequencing of DNA has become more widely used in the management of patients with lung adenocarcinoma; however, no clear mitogenic driver alteration is found in some cases. We evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions and MET exon 14 (METex14) alterations in DNA sequencing (DNAseq) driver–negative lung cancers.

Experimental Design: Lung cancers driver negative by MSK-IMPACT underwent further analysis using a custom RNAseq panel (MSK-Fusion). Tumor mutation burden (TMB) was assessed as a potential prioritization criterion for targeted RNAseq.

Results: As part of prospective clinical genomic testing, we profiled 2,522 lung adenocarcinomas using MSK-IMPACT, which identified 195 (7.7%) fusions and 119 (4.7%) METex14 alterations. Among 275 driver-negative cases with available tissue, 254 (92%) had sufficient material for RNAseq. A previously undetected alteration was identified in 14% (36/254) of cases, 33 of which were actionable (27 in-frame fusions, 6 METex14). Of these 33 patients, 10 then received matched targeted therapy, which achieved clinical benefit in 8 (80%). In the 32% (81/254) of DNAseq driver–negative cases with low TMB [0–5 mutations/Megabase (mut/Mb)], 25 (31%) were positive for previously undetected gene fusions on RNAseq, whereas, in 151 cases with TMB >5 mut/Mb, only 7% were positive for fusions (P < 0.0001).

Conclusions: Targeted RNAseq assays should be used in all cases that appear driver negative by DNAseq assays to ensure comprehensive detection of actionable gene rearrangements. Furthermore, we observed a significant enrichment for fusions in DNAseq driver–negative samples with low TMB, supporting the prioritization of such cases for additional RNAseq.

Translational Relevance

Inhibitors targeting kinase fusions have shown dramatic and durable responses in lung cancer patients, making their comprehensive detection critical. Here, we evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions in patients where no clear mitogenic driver alteration is found by DNA sequencing (DNAseq)–based panel testing. We found actionable alterations (kinase fusions or MET exon 14 skipping) in 13% of cases apparently driver negative by previous DNAseq testing. Among the driver-negative samples tested by RNAseq, those with low tumor mutation burden (TMB) were significantly enriched for gene fusions when compared with the ones with higher TMB. In a clinical setting, such patients should be prioritized for RNAseq. Thus, a rational, algorithmic approach to the use of targeted RNA-based next-generation sequencing (NGS) to complement large panel DNA-based NGS testing can be highly effective in comprehensively uncovering targetable gene fusions or oncogenic isoforms not just in lung cancer but also more generally across different tumor types.

A Commentary is in the same issue at https://clincancerres.aacrjournals.org/content/25/15/4586?iss=15

Wake Up and Smell the Fusions: Single-Modality Molecular Testing Misses Drivers

by Kurtis D. Davies and Dara L. Aisner

Abstract

Multitarget assays have become common in clinical molecular diagnostic laboratories. However, all assays, no matter how well designed, have inherent gaps due to technical and biological limitations. In some clinical cases, testing by multiple methodologies is needed to address these gaps and ensure the most accurate molecular diagnoses.

See related article by Benayed et al., p. 4712

In this issue of Clinical Cancer Research, Benayed and colleagues illustrate the growing need to consider multiple molecular testing methodologies for certain clinical specimens (1). The rapidly expanding list of actionable molecular alterations across cancer types has resulted in the wide adoption of multitarget testing approaches, particularly those based on next-generation sequencing (NGS). NGS-based assays are commonly viewed as “one-stop shops” to detect a vast array of molecular variants. However, as Benayed and colleagues discuss, even well-designed and highly vetted NGS assays have inherent gaps that, under certain circumstances, are ideally addressed by analyzing the sample using an alternative approach.

In the article, the authors examined a cohort of lung adenocarcinoma patient samples that had been deemed “driver- negative” via MSK-IMPACT, an FDA-cleared test that is widely considered by experts in the field to be one of the best examples of a DNA-based large gene panel NGS assay (2). Of 589 driver-negative cases, 254 had additional material amenable for a different approach: RNA-based NGS designed specifically for gene fusion and oncogenic gene isoform detection. After accounting for quality control failures, 232 samples were successfully sequenced, and, among these, 36 samples (representing an astonishing 15.5% of tested cases) were found to be positive for a driver gene fusion or oncogenic isoform that had not been detected by DNA-based NGS. The real-world value derived from this orthogonal testing schema was more than theoretical, with 8 of 10 (80%) patients demonstrating clinical benefit when treated according to the alteration identified via the RNA-based approach.

To detect gene rearrangements that lead to oncogenic gene fusions (and to detect mutations and insertions/deletions that lead to MET exon 14 skipping), MSK-IMPACT employs hybrid capture-based enrichment of selected intronic regions from genomic DNA. While this approach has proven to be successful in a variety of settings, there are associated limitations that were determined in this study to underlie the discrepancies between MSK-IMPACT and the RNA-based assay. First, some introns that are involved in clinically actionable rearrangement events are very large, thus requiring substantial sequencing capital that can represent a disproportionate fraction of the assay. Despite the ability via NGS to perform sequencing at a large scale, this sequencing capacity is still finite, and thus decisions must be made to sacrifice coverage of certain large genomic regions to ensure sufficient sequencing depth for other desired genomic targets. In the case of MSK-IMPACT (and most other DNA-based NGS assays), certain important introns in NTRK3 and NRG1 are not included in covered content, simply because they are too large (>90 Kb each). The second primary problem with DNA-based analysis of introns is that they often contain highly repetitive elements that are extremely difficult to assess via NGS due to their recurring presence across the genome. Attempts to sequence these regions are largely unfruitful because any sequencing data obtained cannot be specifically aligned/mapped to the desired targeted region of the genome (3). This is particularly true for intron 31 of ROS1, because it contains two repetitive long interspersed nuclear elements, and many DNA-based assays, including MSK-IMPACT, poorly cover this intron (4). In this study by Benayed and colleagues, the most common discrepant alteration was fusion involving ROS1, which accounted for 10 of 36 (28%) cases. At least six of these, those that demonstrated fusion to ROS1 exon 32, were likely directly explained by incomplete intron 31 sequencing. RNA-based analysis is able to overcome the above described limitations owing to the simple fact that sequencing is focused on exons post-splicing and the need to sequence introns is entirely avoided (Fig. 1).

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Figure 1.

Schematic representation of underlying genomic complexities that can lead to false-negative gene fusion results in DNA-based NGS analysis. In some cases, RNA-based approaches may overcome the limitations of DNA-based testing.

Lack of sufficient intronic coverage could not account for all of the discrepancies between DNA-based and RNA-based analysis however. Six samples in the cohort were found to be positive for MET exon 14 skipping based on RNA. In five of these, genomic alterations in MET introns 13 or 14 were observed, however they did not conform to canonical splice site alterations and thus were not initially called (although this was addressed by bioinformatics updates). In RNA-based testing, however, determination of exon skipping is simplified such that, regardless of the specific genomic alteration that interferes with splicing, absence of the exon in the transcript is directly observed (5). In another two of the discrepant cases, tumor purity was observed to be low in the sample, meaning that the expected variant allele frequency (VAF) for a genomic event would also likely be low, potentially below detectable levels. However, overexpression of the fusions at the transcript level was theorized to compensate for low VAF (Fig. 1). Additional explanations for discordant findings between the assays included sample-specific poor sequencing in selected introns and complex rearrangements that hindered proper capture (Fig. 1).

The take home message from Benayed and colleagues is simply this: there is no perfect assay that will detect 100% of the potential actionable alterations in patient samples. Even an extremely well designed, thoroughly vetted, and FDA-cleared assay such as MSK-IMPACT will have inherent and unavoidable “holes” due to intrinsic limitations. The solution to this dilemma, as adeptly described by Benayed and colleagues, is additional testing using a different approach. While in an ideal world every clinical tumor sample would be tested by multiple modalities to ensure the most comprehensive clinical assessment, the reality is that these samples are often scant and testing is fiscally burdensome (and often not reimbursed). Therefore, algorithms to determine which samples should be reflexed to secondary assays after testing with a primary assay are critical for maximizing benefit. In this study, the first algorithmic step was lack of an identified driver (because activated oncogenic drivers tend to exist exclusively of each other), which amounted to 23% of samples tested with the primary assay. In addition, the authors found a significantly higher rate of actionable gene fusions in samples with a low (<5 mut/Mb) tumor mutational burden, meaning that this metric, which was derived from the primary assay, could also be used to help inform decision making regarding additional testing. While this scenario is somewhat specific to lung cancer, similar approaches could be prescribed on a cancer type–specific basis.

These findings should be considered a “wake-up call” for oncologists in regard to the ordering and interpretation of molecular testing. It is clear from these and other published findings that advanced molecular analysis has limitations that require nuanced technical understanding. As this arena evolves, it is critical for oncologists (and trainees) to gain an increased comprehension of how to identify when the “gaps” in a test might be most clinically relevant. This requires a level of technical cognizance that has been previously unexpected of clinical practitioners, yet is underscored by the reality that opportunities for effective targeted therapy can and will be missed if the treating oncologist is unaware of how to best identify patients for whom additional testing is warranted. This study also highlights the mantra of “no test is perfect” regardless of prestige of the testing institution, number of past tests performed, or regulatory status. NGS, despite its benefits, does not mean all-encompassing. It is only through the adaptability of laboratories to utilize knowledge such as is provided by Benayed and colleagues that advances in laboratory medicine can be quickly deployed to maximize benefits for oncology patients.

References:

1. Comprehensive genomic characterization of squamous cell lung cancers. Nature 2012, 489(7417):519-525.
2. A genomics-based classification of human lung tumors. Science translational medicine 2013, 5(209):209ra153.
3. Govindan R, Ding L, Griffith M, Subramanian J, Dees ND, Kanchi KL, Maher CA, Fulton R, Fulton L, Wallis J et al: Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 2012, 150(6):1121-1134.
4. Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, Asaka R, Hamanaka W, Ninomiya H, Uehara H et al: RET, ROS1 and ALK fusions in lung cancer. Nature medicine 2012, 18(3):378-381.
5. Morodomi Y, Takenoyama M, Inamasu E, Toyozawa R, Kojo M, Toyokawa G, Shiraishi Y, Takenaka T, Hirai F, Yamaguchi M et al: Non-small cell lung cancer patients with EML4-ALK fusion gene are insensitive to cytotoxic chemotherapy. Anticancer research 2014, 34(7):3825-3830.
6. Yoshimura M, Tada Y, Ofuzi K, Yamamoto M, Nakatsura T: Identification of a novel HLA-A 02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene. Oncology reports 2014, 32(1):33-39.
7. Yang L, Li G, Zhao L, Pan F, Qiang J, Han S: Blocking the PI3K pathway enhances the efficacy of ALK-targeted therapy in EML4-ALK-positive nonsmall-cell lung cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2014.
8. Workman P, van Montfort R: EML4-ALK fusions: propelling cancer but creating exploitable chaperone dependence. Cancer discovery 2014, 4(6):642-645.
9. Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E, Cho J, Suh J, Capelletti M, Sivachenko A et al: Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 2012, 150(6):1107-1120.
10. Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, Plenker D, Leenders F, Sun R, Zander T et al: Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nature genetics 2012, 44(10):1104-1110.

Other posts on this site which refer to Lung Cancer and Cancer Genome Sequencing include:

Multi-drug, Multi-arm, Biomarker-driven Clinical Trial for patients with Squamous Cell Carcinoma called the Lung Cancer Master Protocol, or Lung-MAP launched by NCI, Foundation Medicine, and Five Pharma Firms

US Personalized Cancer Genome Sequencing Market Outlook 2018 –

Comprehensive Genomic Characterization of Squamous Cell Lung Cancers

International Cancer Genome Consortium Website has 71 Committed Cancer Genome Projects Ongoing

Non-small Cell Lung Cancer drugs – where does the Future lie?

Lung cancer breathalyzer trialed in the UK

Diagnosing Lung Cancer in Exhaled Breath using Gold Nanoparticles

Multi-drug, Multi-arm, Biomarker-driven Clinical Trial for patients with Squamous Cell Carcinoma called the Lung Cancer Master Protocol, or Lung-MAP launched by NCI, Foundation Medicine, and Five Pharma Firms

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State of the art in oncologic imaging of lungs.

Posted in Biomarkers & Medical Diagnostics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Health Economics and Outcomes Research, HealthCare IT, Imaging-based Cancer Patient Management, Medical Devices R&D and Inventions, Medical Devices R&D Investment, Medical Imaging Technology, Image Processing/Computing, MRI, CT, Nuclear Medicine, Ultra Sound, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Investment, Population Health Management, Genetics & Pharmaceutical, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Uncategorized, tagged American Cancer Society, Cancer - General, cancer deaths, cancer imaging, cancer mortality, Clinical trial, CT, FDA, health, lung, lung adenocarcinoma, Lung cancer, medicine, National Institutes of Health, NCI, Non-small cell lung cancer, PET, PET/CT, relative survival rate, research, science on January 23, 2013| 5 Comments »

State of the art in oncologic imaging of lungs.

Author and Curator: Dror Nir, PhD

• 

This is the second post in a series in which I will address the state of the art in oncologic imaging based on a review paper; Advances in oncologic imaging^†‡ that provides updates on the latest approaches to imaging of 5 common cancers: breast, lung, prostate, colorectal cancers, and lymphoma. This paper is published at CA Cancer J Clin 2012. © 2012 American Cancer Society.

The paper gives a fair description of the use of imaging in interventional oncology based on literature review of more than 200 peer-reviewed publications.

In this post I summaries the chapter on lung cancer imaging.

Lung Cancer Imaging

“Lung cancer remains the most common cause of death from cancer worldwide, having resulted in 1.38 million deaths (18.2% of all cancer deaths) in 2008.48 It also represents the leading cause of death in smokers and the leading cause of cancer mortality in men and women in the United States. In 2012, it was estimated that 226,160 new cases of lung cancer would be diagnosed (accounting for about 14% of cancer diagnoses) and that lung cancer would cause 160,340 deaths (about 29% of cancer deaths in men and 26% of cancer deaths in women) in the United States.1 The 1-year relative survival rate for the disease increased from 35% to 43% from 1975 through 1979 to 2003 through 2006.49 The 5-year survival rate is 53% for disease that is localized when first detected, but only 15% of lung cancers are diagnosed at this early stage.”

For cancer with such poor survival rates removal of the primary lesion by surgery at an early-stage disease is the best option. The current perception in regards to lung cancr is that patients may have subclinical disease for years before presentation. It is also known that early lung cancer lesions; adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) are slow-growing, doubling time which can exceed 2 years.52 But, since at present, no lung cancer early-detection biomarker is clinically available, the diagnosis of this disease is primarily based on symptoms, and detection often occurs after curative intervention and when it’s already too late – see: Update on biomarkers for the detection of lung cancer and also Diagnosing lung cancer in exhaled breath using gold nanoparticles. Until biomarker is found, the burden of screening for this disease is on imaging.

“AIS and MIA generally appear as a single peripheral ground-glass nodule on CT. A small solid component may be present if areas of alveolar collapse or fibroblastic proliferation are present,50, 51 but any solid component should raise concern for a more invasive lesion (Fig. 8). Growth over time on imaging can often be difficult to assess due to the long doubling time of these AIS and MIA, which can exceed 2 years.52 However, indicators other than growth, such as air bronchograms, increasing density, and pleural retraction within a ground-glass nodule are suggestive of AIS or MIA.

CT image shows a ground glass nodule, which is the typical appearance of AIS, in the right upper lobe.

 

CT (A) demonstrated extensive consolidation with air bronchograms in the left upper lobe, which at surgical resection were found to represent adenocarcinoma of mixed subtype with predominate (70%) mucinous bronchioloalveolar subtype. PET imaging in the same patient (B) demonstrated uptake in the lingula higher than expected for bronchioloalveolar carcinoma and probably due to secondary inflammation/infection. CT (C) obtained 3 years after images (A) and (B) demonstrated biopsy-proven recurrent soft-tissue mass near surgical site. Fused FDG/PET images (D) demonstrate no uptake in the area. This finding is consistent with the decreased uptake usually seen in tumors of bronchioloalveolar histology (new terminology of MIA).

In August 2011 the results of the “National Lung Screening Trial “ which was funded by the National Cancer Institute (NCI) were published in NEJM; Reduced Lung-Cancer Mortality with Low-Dose Computed Tomographic Screening. This randomized study results showed that with low-dose CT screening of high-risk persons, there was a significant reduction of 20% in the mortality rate from lung cancer as compared to chest radiographs screening.

Based on these results one can find the following information regarding Lung Cancer Screening on the NCI web-site:

“Three screening tests have been studied to see if they decrease the risk of dying from lung cancer.

The following screening tests have been studied to see if they decrease the risk of dying from lung cancer:

• Chest x-ray: An x-ray of the organs and bones inside the chest. An x-ray is a type of energy beam that can go through the body and onto film, making a picture of areas inside the body.
• Sputum cytology: Sputum cytology is a procedure in which a sample of sputum (mucus that is coughed up from the lungs) is viewed under a microscope to check for cancer cells.
• Low-dose spiral CT scan (LDCT scan): A procedure that uses low-dose radiation to make a series of very detailed pictures of areas inside the body. It uses an x-ray machine that scans the body in a spiral path. The pictures are made by a computer linked to the x-ray machine. This procedure is also called a low-dose helical CT scan.

Screening with low-dose spiral CT scans has been shown to decrease the risk of dying from lung cancer in heavy smokers.

A lung cancer screening trial studied people aged 55 years to 74 years who had smoked at least 1 pack of cigarettes per day for 30 years or more. Heavy smokers who had quit smoking within the past 15 years were also studied. The trial used chest x-rays or low-dose spiral CT scans (LDCT) scans to check for signs of lung cancer.

LDCT scans were better than chest x-rays at finding early-stage lung cancer. Screening with LDCT also decreased the risk of dying from lung cancer in current and former heavy smokers.

A Guide is available for patients and doctors to learn more about the benefits and harms of low-dose helical CT screening for lung cancer.

Screening with chest x-rays or sputum cytology does not decrease the risk of dying from lung cancer.

Chest x-ray and sputum cytology are two screening tests that have been used to check for signs of lung cancer. Screening with chest x-ray, sputum cytology, or both of these tests does not decrease the risk of dying from lung cancer.”

The authors of Advances in oncologic imaging^†‡ found out that for pre-treatment staging and post treatment follow-up of lung cancer patients mainly involves CT (preferably contrast enhanced, FDG PET and PET/CT. “Integrated PET/CT has been found to be more accurate than PET alone, CT alone, or visual correlation of PET and CT for staging NSCLC (Non-small-cell lung carcinoma).59 “

The standard treatment of choice for localized disease remains surgical resection with or without chemo-radiation therapy (stage dependant). “The current recommendations for routine follow-up after complete resection of NSCLC are as follows: for 2 years following surgery a contrast-enhanced chest CT scan every 4 to 6 months and then yearly non-contrast chest CT scans.62 Detection of recurrence on CT is the primary goal in the initial years, and therefore, optimally, a contrast-enhanced scan should be obtained to evaluate the mediastinum. In subsequent years, when identifying an early second primary lung cancer becomes of more clinical importance, a non-contrast CT chest scan suffices to evaluate the lung parenchyma.

CT (A) of 78-year-old male who was status post–left lobe lobectomy and left upper lobe wedge resection shows recurrent nodule at the surgical resection site. Fused PET/CT (B) demonstrates increased [18F]FDG uptake in the corresponding nodule at the surgical resection site consistent with recurrent tumor.

In patients undergoing chemotherapies: “ [¹⁸F]FDG PET response correlates with histologic response.63 [¹⁸F]FDG PET scan data can provide an early readout of response to chemotherapy in patients with advanced-stage lung cancer.64

In patients treated by recently developed “Targeted Therapies” such as Radiofrequency ablation (RFA) the authors found out that PET/CT is the preferred imaging modality for post treatment follow-up.

“ Most patients treated with pulmonary ablation will have had a pre-procedure CT or a fusion PET/CT scan, which allows more precise anatomic localization of abnormalities seen on PET. Generally, either CT or PET/CT is performed within a few weeks of the procedure to provide a new baseline to which future images can be compared to assess for changes in size, degree of enhancement or [¹⁸F]FDG avidity.67”

CT (A) demonstrates new left upper lobe mass representing new primary NSCLC in a patient who had a status post–right pneumonectomy for a prior NSCLC. CT (B) obtained in the same patient 2 weeks after radiofrequency ablation (RFA) demonstrates the postablation density in the left upper lobe. Fused PET/CT (C) obtained 4 months after RFA demonstrates mild [18F]FDG uptake at RFA site in the left upper lobe consistent with posttreatment inflammation. Fused PET/CT (D) obtained 7 months after RFA demonstrates new focal [18F]FDG uptake at post-RFA-opacity consistent with recurrent tumor.

Prostate Cancer Imaging

To be followed…

Other research papers related to the management of Lung cancer were published on this Scientific Web site:

Diagnosing lung cancer in exhaled breath using gold nanoparticles

Lung Cancer (NSCLC), drug administration and nanotechnology

Non-small Cell Lung Cancer drugs – where does the Future lie?

Comprehensive Genomic Characterization of Squamous Cell Lung Cancers

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