A Moving Picture

In the previous blog we caught up with the ongoing lung TRACERx study, specifically its work on the effect of air-born pollutants. However, TRACERx has a much broader overall aim, namely to follow the evolution of human lung cancers over time — tracking patterns of cell lineages (clones) of tumour cells, monitoring gene expression, tracking circulating tumour DNA (ctDNA) in the bloodstream and defining how immune cells target lung cancer (see figure below).

It’s important because lung cancer is the leading global cause of cancer-related deaths. The  two subtypes are non-small cell lung cancer (NSCLC: 85% of cases), and small cell lung cancer (the remaining 15%). NSCLC comes in three subtypes based on what the cells look like (lung adenocarcinoma, lung squamous cell carcinoma and large cell carcinoma).

The scale of these studies is hard to grasp for they examined NSCLC in 421 individuals and did genomic profiles of 1,644 tumour regions.

The moving picture of lung cancer. The scheme outlines the TRACERx project: tumour samples are analysed for patterns of cell lineages (clones) by comparing DNA and RNA sequences, for immune cells in the tumour microenvironment and for changes in tumour cells that have spread (metastasized) to distant sites. Circulating tumour DNA (ctDNA) shed into the bloodstream was also screened to monitor disease progression and response to treatment. From Hayes and Meyerson 2023.

The main aim

The central idea behind TRACERx is to map how the variables summarised above relate to clinical outcome — i.e. disease-free survival, the survival time after treatment without cancer symptoms. The approach is to get a view of the constantly changing picture of tumour heterogeneity by sequencing the protein-coding regions of known cancer driving genes (by whole-exome sequencing or by sequencing the whole-genome). This should reveal the effects of chemotherapy (e.g., platinum-based drugs).

In addition to the air-born pollutant work, the consortium has just published four more papers that reflect progress towards these aims. For the purposes of this blog we will attempt a brief summary of these — with sincere apologies to those whose fantastic work has thus been subsumed.

Jargon buster

First a note about the terminology of mutations in cancer:

(i) clonal mutations are shared by all cancer cells.

(ii) subclonal mutations are present only in a subset of tumour cells. A subclone is a descendant of the most recent common ancestor (MRCA) of the tumour sample.

(iii) truncal mutations are mutations present in the trunk of the cancer evolutionary tree, i.e. mutations present in all subclones and at all timepoints.

(iv) Whole-genome doubling (WGD) involves doubling of the entire chromosome complement. It is a prevalent event in cancer.

(v) A clonal sweep occurs when a subclone outcompetes its neighboring cells, resulting in a trend of reduced diversity and towards a homogeneous tumour.

Key points

1. Lung adenocarcinoma: Frankell et al. found sub-clonal mutations in 22 of 40 common ‘cancer genes’. These included TP53 and KRAS. Truncal mutations in TP53 and KRAS tended to be mutually exclusive and these were also exclusive for EGFR whole genome doubling (WGD). Amplification of MYC and activating mutations in receptor tyrosine kinase pathways (both major cancer drivers) were truncal events, in contrast to the generally subclonal events affecting TP53 and KRAS, despite the latter being major cancer drivers.

Subclonal WGD was detected in 19% of tumours and 10% of tumours had multiple subclonal WGDs. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Notably, 8% of these tumours in smokers showed no evidence of tobacco-induced mutations but they carried patterns of mutations in EGFR and other oncogenes (RET, ROS1, ALK and MET) resembling those found in never-smokers, suggesting similar causes.

In 1% of cases lung tumours were observed that had two distinct genomic origins. Called ‘collision tumours’ these consist of two distinct tumours that have grown to occupy the same region of the lung as a single, continuous mass.

Overall these results show the importance of clonal expansion, WGD and copy number instability in regulating the behaviour of non-small cell lung cancer.

2. Metastasis: Al Bakir et al. compared the genomes (DNA sequences) of primary tumours with those of their metastases and found that in 25% of cases metastatic growth diverged early, before the final clonal sweep in the primary. This shows up as a set of mutations in all regions of the primary that was absent from metastases. Early divergence often occurred in small tumours (less than 8 mm diameter) and was more common in smokers. All of which highlights the current limitations of radiological screening for identifying early diverging tumours and the problems of targeting metastasis-seeding subclones.

3. Metastasis: Abbosh et al. developed a bioinformatics method (ECLIPSE) to track low levels of ctDNA and thus identify cases of polyclonal metastatic dissemination that are associated with poor outcome. They also showed that ctDNA could forecast impending relapse and that it is useful for selecting patients who might benefit from drug treatments.

4. Intra-tumour heterogeneity: Martínez-Ruiz et al. looked at gene expression (i.e. the RNA molecules being made {transcribed} at any time) in non-small cell lung tumours and found that the outgrowth of a clone containing a specific mutation was positively selected when that gene was highly expressed. In addition they looked at different versions (alleles) of the same gene and found that both copy-number-dependent and copy-number-independent events could affect the expression of an allele (gene copy number means the number of copies of a given gene in an organism’s complete set of genes).

It emerged that copy-number-independent events affected epigenetic regulators (i.e. DNA and histone modulators). In particular mutations in a set of epigenetic modifiers (CREBBP, KDM5C, SMARCA4, SETD2 and KMT2B) were associated with increased levels of copy-number-independent alleles. By contrast, mutations in the de-methylase KDM6A were associated with decreased expression of these alleles.

These summaries do no justice at all to the huge amount of work that has produced these papers. Far from answering all the problems of lung cancer, they rather highlight our ignorance — but the advances they make on a range of fronts show that the enigma of lung cancer is slowly being prized open.

References

Hayes TK, Meyerson M. Molecular portraits of lung cancer evolution. Nature. 2023 Apr;616(7957):435-436. doi: 10.1038/d41586-023-00934-0. PMID: 37045956.

Frankell, A.M., Dietzen, M., Al Bakir, M. et al. The evolution of lung cancer and impact of subclonal selection in TRACERx. Nature 616, 525–533 (2023). https://doi.org/10.1038/s41586-023-05783-5

Al Bakir, M., Huebner, A., Martínez-Ruiz, C. et al. The evolution of non-small cell lung cancer metastases in TRACERx. Nature 616, 534–542 (2023). https://doi.org/10.1038/s41586-023-05729-x

Abbosh, C., Frankell, A.M., Harrison, T. et al. Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA. Nature 616, 553–562 (2023). https://doi.org/10.1038/s41586-023-05776-4

Martínez-Ruiz, C., Black, J.R.M., Puttick, C. et al. Genomic–transcriptomic evolution in lung cancer and metastasis. Nature 616, 543–552 (2023). https://doi.org/10.1038/s41586-023-05706-4

The child is the father of the man

“The child is the father of the man” observed William Wordsworth — referring to the joy of beholding a rainbow that is undimmed as one grows older. Joy isn’t a sensation much associated with cancer but the master poet’s words came to mind recently on reading a study of childhood cancers by Neekesh Dharia, Todd Golub and colleagues from the Dana-Farber Cancer Institute, Boston and other US centres.

The last 50 years has seen remarkable advances in treatment of cancers in young people such that in high-income countries over 80% of cases are cured, albeit often with serious, long-term side effects. However, these particularly distressing diseases remain a grave problem and globally some 400,000 are diagnosed each year in young people up to the age of nineteen — that’s one every three minutes. In addition, it’s estimated that across the world about one third of child cancers are not diagnosed at all. In the USA about 16,000 children under 19 years of age are diagnosed with cancer each year and, despite treatment advances, some 20% do not survive. In the UK the figures are nearly 2000 new cases and about 240 deaths.

Childhood cancers are unusual

Pediatric cancers are of great interest from a therapeutic point of view because they break the general rule that tumours arise from the accumulation of mutations in DNA — i.e. genetic damage — and, because this takes time, cancers are generally regarded as diseases of old age. In children, however, cancers are frequently driven by a single genetic change and overall they have about one thousand times fewer mutations than many adult cancers. An example of a single driver would be the fusion of two genes (EWSR1 and FLI1) in Ewing sarcoma. This feature raises the question of whether they also differ from adult tumours in requiring fewer genes for the survival and growth of their cells. And that’s important because, if true, it implies that there are fewer targets for therapy.

Neekesh Dharia and colleagues first confirmed that pediatric cancers have low mutational burdens (or ‘quiet’ genomes) compared to those of adult tumours — some 1,000-fold fewer somatic mutations. The left-hand figure below shows the range of mutations in normal cells (below 10 per megabase), the spread in a variety of adult tumours (from 10 to 1000) with pediatric cancers in between (10 to 100 mutations per megabase).

Mutation rates (left) and the number of genes essential for survival. Left: number of mutations per megabase (million bases) of DNA for normal cells, pediatric tumour cells and a range of adult tumour cells. Right: The number of essential genes required to keep pediatric tumour cells and adult tumour cells growing in culture. From Dharia et al. 2021.

Looking for weaknesses

We’ve recently noted that CRISPR-Cas can not only be used to knock out genes or to insert new DNA into genomes (Cardiff Crock of Gold?) but can also be used to scan DNA. The power of this method enabled the Boston group to screen hundreds of adult cancer cell lines together with 178 pediatric cancer cell lines that they assembled. They confirmed that, despite being grown in culture, their cells had not acquired significant additional mutations from those present in the tumours from which they were derived — so they remained acceptable as tumour models. The aim was to knock out in turn each of a panel of genes to determine which were essential for the cells to survive — the idea being, of course, that if you could hit one of those with a blocking drug the cell would die.

The answer is clear in the right-hand figure above: there’s no real difference between pediatric cancers and adult solid tumours in the number of genes required for cell survival. You can view the complete pediatric cancer dependency map on line.

A new range of treatment

This is an important and exciting result because there are drugs available that block the action of the proteins made from some of these genes. Thus in adult cancer lines, activating mutations in ALK and BRAF (encoding kinases) made the cells dependent on these proteins — in other words, drugging ALK or BRAF killed the cells. In another example pediatric cancer cells with normal (wild-type) p53 depended on the regulator MDM2 for survival — pointing to MDM2 inhibitors as a possible therapy.

A surprisingly large proportion of pediatric cancers required the anti-apoptotic protein MCL1 to grow and growth was suppressed by a small molecule inhibitor of MCL1 (S63845).

An immense amount of work went into this paper and it’s gratifying that it has produced some clear-cut pointers. The most important is that screening cancers in children for mutations that can be targetted by drugs in use for adult tumours opens a new therapeutic window.

And that’s important because,  notwithstanding their impact on families, pediatric cancers represent a small commercial market with correspondingly limited investment — so progress in drug development lags behind that for more dominant types of cancer.

Reference

Dharia, N.V., Kugener, G., Guenther, L.M. et al. 2021. A first-generation pediatric cancer dependency map. Nat Genet 53, 529–538. https://doi.org/10.1038/s41588-021-00819-w