Commentary on tumor heterogeneity

I would like to thank all three authors Drs. Ilie and Paul Hofman, and Mari Mino-Kennudson for their state of art overview on the role of liquid biopsy present and in future and the correlation to tissue biopsy (1-4). On this topic it could not be a classical pro&con discussion, as nobody in the pathologic community would neglect liquid biopsy. However, the strategy to what extend liquid biopsy can be integrated into the daily practice in pathology remains to be defined. In that regard we can see slight differences how liquid biopsy will be implemented in the US and in Europe.

As for the costs raised in Dr. Hofman’s article it can be stated, that any kind of pathologic analysis is by far the cheapest diagnostic test among most clinical and radiological tests. And when correlated to the costs of targeted therapy, these costs can only be called “peanuts”. In their analysis tumor heterogeneity either from the genetic point but also from morphology have been stressed as an argument for liquid biopsy. This aspect needs a commentary.

Morphologic heterogeneity of NSCLC is well known, and has been introduced into the classification of lung cancer. Adenocarcinomas are classified according to their predominant pattern, squamous cell carcinomas may be composed of well-differentiated and areas of undifferentiated carcinoma portions. Sarcomatoid carcinomas are characterized by a combination of well-differentiated carcinoma with sarcomatoid looking carcinomas. Adenosquamous carcinoma is composed of two different types of carcinoma. But does that mean these different components are genetically heterogeneous? The answer is yes and no. To bring light into this controversial discussed topic, we will discuss heterogeneity in primary tumors as well as metastasis, before asking, what liquid biopsy can add.

In some studies EGFR, KRAS, and PI3K mutations have been identified in both components of adenosquamous carcinomas (5), EGFR mutations were homogenously distributed in primary lung carcinomas. In contrast ALK rearrangements in the same series showed intra-tumor heterogeneity of rearrangements. In another study analyzing 629 patient tumor samples 9 out of 30 carcinomas with ALK rearrangements showed heterogeneity in the fusion transcript, whereas no heterogeneity was seen in 364 samples with EGFR mutations. Interestingly ALK fusions were positively associated with a micropapillary pattern and negatively associated with a lepidic pattern pointing to some morphologic and genetic associations (6). Even in sarcomatoid carcinomas, which present with heterogeneity of morphological patterns the concordance rate for mutations was relatively high with 61% (7). Similarly samples taken from different areas of surgically removed carcinomas showed a high degree of homogeneous mutational profiles for EGFR and KRAS (18 of 19 cases) (8). There was only one study showing a significant intra-tumoral heterogeneity. However, the authors did not differentiate driver mutations and other mutations. So they concluded heterogeneity on the basis of multiple mutations in subclones within the primary tumor (9). Additional mutations might reflect adaptations of subclones to hypoxia, changes in metabolism. This aspect is elucidated in the study by de Bruin, which sequenced 25 spatially distinct regions from seven operable NSCLCs and found evidence of branched evolution, with driver mutations arising before and after subclonal diversification. There was pronounced intra-tumor heterogeneity in copy number alterations, translocations, and mutations associated with metabolic enzyme activity (10). This point to individual pathways of carcinogenesis, with either homogeneous or heterogeneous types of driver mutations.

What about heterogeneity in metastasis: When looking up primary tumor and metastasis, some studies showed the same type of mutation in both, whereas in other studies subclones do exist in the primary as well as in the metastatic site, which are genetically different.

In a review summarizing 26 different published studies a substantial concordance was observed between primary and metastatic tumors in terms of EGFR, KRAS, BRAF, p16 and p53 mutations. However, some level of discordance was seen in most studies; testing methodologies appeared to play a key role in this, along with underlying tumor heterogeneity (11). In the study by Chang et al. p53 and EGFR mutation/overexpression status was different between primary tumors and lymph node metastases in only 5.4/7.2% and 28.6/33.9%, respectively. In most cases, the p53 and EGFR mutations usually preceded lymph node metastasis, and these gene statuses in the primary cancer and their lymph node metastasis were concordant (92.9% and 69.6%). Therefore when p53 mutations occur before the establishment of lymph node metastasis, they subsequently persist in the metastatic nodes (12).

In a series of 30 EGFR mutated adenocarcinomas the mutation was detected in 28 lymph node metastases. In 12 cases there were discordant EGFR mutations between primary tumors and metastasis. In 11 cases EGFR mutations were detected only in the primary tumor, whereas in 1 case only in lymph node metastases (13). In the study by Kim et al. 41 primary tumor and matched metastatic lymph nodes were analyzed by next generation sequencing. Two hundred and thirteen non-synonymous mutations, 32 deletions, and four insertions were discovered. Non-synonymous mutations were seen more often in the primary tumors when the mutation profiles between primary tumor and metastatic L/N were compared, 13 (31.7%) of 41 cases showed discrepant mutation profiles (14).

Finally the genetic makeup of carcinomas can change under the pressure of tyrosine kinase inhibitor (TKI) therapy. In some cases a minor subclone does exist, which contain a resistance mutation in addition to the driver gene mutation (Del exon 19 + T790M mutation) (15), but there are also secondary somatic mutations arising under the pressure of targeted therapy (16,17). This happens in the EGFR gene as well as in the ALK and ROS1 genes. And the trans-differentiation of an adenocarcinoma into a small cell carcinoma under targeted therapy seems to be preceded by a loss of RB and mutations in TP53, somatic mutations, which are not present in the original tumor.

When dissecting the process of invasion and metastasis additional modifications of the genetic machinery is required to communicate with the environment, avoid attack by the immune system, and many more aspects (18). This gives rise to genetic and epigenetic modifications, which I avoid to call heterogeneity. Even within the primary tumor different metabolic requirements might result in genetic/epigenetic modifications: Tumor cells in the center are exposed to hypoxia and acidic pH, whereas tumor cells in the periphery are better supplied by oxygen and nutrients. Tumor cells at the periphery are more exposed to stroma cells, and have to communicate with these cells, a task, which tumor cells in the center portion might not need. These are all adaptations in addition to the primary genetic changes driving carcinogenesis.

When coming to circulating tumor DNA (cfDNA) the question of tumor heterogeneity has not been extensively addressed. In comparing tissue biopsies and cfDNA in most cases there is concordant finding of the same mutations in both. However, there are samples with discordant findings: mutated cfDNA and wild type tissue DNA and the reverse. In the case of biopsies the explanation is usually the small size of the tissue, explaining a negative finding. But analysis of cfDNA so far is limited as in most studies single genes are analyzed, whereas the discussion on heterogeneity in tumors is based on the analysis of multiple genes. Therefore let us await more data on next generation sequencing using plasma samples, before we can consider that cfDNA analysis might be useful to pick up heterogeneous tumor cells clones.

Acknowledgements

None.

Footnote

Conflicts of Interest: The author has no conflicts of interest to declare.

Comment on: Ilié M, Hofman P. Pros: Can tissue biopsy be replaced by liquid biopsy? Transl Lung Cancer Res 2016;5:420-3.
Mino-Kenudson M. Cons: Can liquid biopsy replace tissue biopsy?—the US experience. Transl Lung Cancer Res 2016;5:424-7.
Ilié M, Hofman P. Rebuttal from Dr. Hofman and Dr. Ilié. Transl Lung Cancer Res 2016;5:428-9.
Mino-Kenudson M. Rebuttal from Dr. Mino-Kenudson. Transl Lung Cancer Res 2016;5:430-2.

References

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3. Ilié M, Hofman P. Rebuttal from Dr. Hofman and Dr. Ilié. Transl Lung Cancer Res 2016;5:428-9.
4. Mino-Kenudson M. Rebuttal from Dr. Mino-Kenudson. Transl Lung Cancer Res 2016;5:430-2.
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