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Page 4 of 19 Shi et al. J Cancer Metastasis Treat 2018;4:47 I http://dx.doi.org/10.20517/2394-4722.2018.32

Cellular composition of tumors
Cell types present in the tumor stroma, such as immune cells, fibroblasts, vascular cells play a critical role
in shaping the composition of tumors by secreting cytokines growth factors and extracellular matrix that
[23]
changes the stiffness of the tumor tissue . In a tumor microenvironment infiltrated by CD8 T cells at the
tumor site is associated with increased overall survival, whereas myeloid-derived suppressor cells (MDSCs)
[24]
possessing strong immune suppressive activity decreases overall survival . The diversity of these func-
tionally different immune cell types creates a heterogeneous tumor microenvironment and regulate tumor
[25]
growth, metastasis and treatment response . In addition, the distribution and density of the vasculature
impact the supply of nutrients and oxygen selecting for tumor cells with specific metabolic phenotypes fur-
ther contributing to tumor heterogeneity [26,27] . Tumor heterogeneity has a significant bearing on the manage-
ment of disease as summarized in the next section.

CLINICAL IMPACT OF THE ITH
Resistance to therapy
The resistance of tumors to therapies is often attributed to the presence of rare drug-resistant clones in the
tumor before therapy or appears after treatment. An example of clonal resistance was observed in patients
with anaplastic lymphoma kinase (ALK gene) rearranged non-small cell lung cancer (NSCLC) post treat-
[28]
ment with ALK inhibitors . Patients that developed drug resistance displayed a distinct spectrum of ALK
[28]
resistance mutations in response to different generations of ALK inhibitors . Particularly, ALK G1202R muta-
tion is highly enriched in resistant tumors after treatment with second-generation ALK inhibitors, highlight-
[28]
ing the significance of repeat biopsies and genotyping during the course of targeted therapy treatment .
In addition, studies investigating the mechanism of resistance of NSCLC tumors to EGFR tyrosine kinase
inhibitors have revealed a variety of drug resistance mechanisms, including gatekeeper mutation T790M
[29]
[30]
detected in > 50% of the EGFR TKI resistant tumors , amplification of MET receptor tyrosine kinase ,
[31]
activating mutation in PI3K pathway , and other uncharacterized mechanisms involving changes in the
cellular phenotype. The appearance of a rare clonal population of tumor cells harboring drug resistance
mutations or drug resistance phenotype can be captured by single-cell sequencing of the tumor and may
not be discernible from whole tumor analysis, especially when present at a very low frequency. In an alter-
native model of drug resistance, resistant clones can be pre-existing in the tumor as a rare cell population
and emerge post clearance of the drug-susceptible clones. In fact, in a study involving a cohort of 20 breast
cancer patients, 8 out of 10 patients that did not show complete clearance of the tumor displayed unique so-
matic mutations in chemoresistant clones by single-cell sequencing. These mutations were pre-existing and
[32]
were adaptively selected by the chemotherapy treatment . It is possible to detect de novo or drug-induced
resistant clones present at low frequency by ultra-deep exome sequencing, however, two critical pieces of in-
formation - number of cells harboring the mutation and the zygosity of the mutation - cannot be accurately
assessed from the bulk sequencing.

Challenges in diagnostic and prognostic biomarker identification
Identifying clinically relevant diagnostic biomarkers are challenging given that the tumor is heterogeneous
and diagnostic or prognostic biomarkers are not expressed uniformly in all cells and across longitudinal
assessment periods [Figure 3]. For example, the divergent genetic landscape of metastatic cells can render
[33]
biomarkers identified from primary tumors irrelevant [Figure 3] .
In prostate cancer, ITH represents a major challenge for diagnostic and prognostic biomarker identifica-
tion. Enhanced DNA ploidy and loss of PTEN, a tumor-suppressor gene, are critical prognostic markers of
[34]
prostate cancer . In a clinical study of 304 patients who underwent radical prostatectomy, a significant dif-
ference in DNA ploidy classification and loss of PTEN expression was observed by analyzing all tumor areas
in comparison to a single biopsy sample, suggesting that the heterogeneous chromosomal alterations com-