Ado-trastuzumab emtansine (T-DM1) in patients with HER2-amplified tumors excluding breast and gastric/gastroesophageal junction (GEJ) adenocarcinomas: results from the NCI-MATCH trial (EAY131) subprotocol Q.

BACKGROUND: The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) is a national precision medicine study incorporating centralized genomic testing to direct refractory cancer patients to molecularly targeted treatment subprotocols. This treatment subprotocol was designed to screen for potential signals of efficacy of ado-trastuzumab emtansine (T-DM1) in HER2-amplified histologies other than breast and gastroesophageal tumors. METHODS: Eligible patients had HER2 amplification at a copy number (CN) >7 based on targeted next-generation sequencing (NGS) with a custom Oncomine AmpliSeq™ (ThermoFisher Scientific) panel. Patients with prior trastuzumab, pertuzumab or T-DM1 treatment were excluded. Patients received T-DM1 at 3.6 mg/kg i.v. every 3 weeks until toxicity or disease progression. Tumor assessments occurred every three cycles. The primary end point was centrally assessed objective response rate (ORR). Exploratory end points included correlating response with HER2 CN by NGS. The impact of co-occurring genomic alterations and PTEN loss by immunohistochemistry were also assessed. RESULTS: Thirty-eight patients were enrolled and 36 included in efficacy analysis. Median prior therapies in the metastatic setting was 3 (range 0-9; unknown in one patient). Median HER2 CN was 17 (range 7-139). Partial responses were observed in two (5.6%) patients: one mucoepidermoid carcinoma of parotid gland and one parotid gland squamous cell cancer. Seventeen patients (47%) had stable disease including 8/10 (80%) with ovarian and uterine carcinomas, with median duration of 4.6 months. The 6-month progression-free survival rate was 23.6% [90% confidence interval 14.2% to 39.2%]. Common toxicities included fatigue, anemia, fever and thrombocytopenia with no new safety signals. There was a trend for tumor shrinkage with higher levels of gene CN as determined by the NGS assay. CONCLUSION: T-DM1 was well tolerated. While this subprotocol did not meet the primary end point for ORR in this heavily pre-treated diverse patient population, clinical activity was seen in salivary gland tumors warranting further study in this tumor type in dedicated trials.

Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon.

We report that CpG island methylation, an epigenetic modification of DNA known to correlate closely with silencing of gene transcription, appears in the oestrogen receptor (ER) gene in a subpopulation of cells which increases as a direct function of age in human colonic mucosa. This same methylation change characterizes virtually all cells in all 45 colorectal tumours examined, including the earliest stages of tumour formation. ER gene expression is diminished or absent in colorectal tumours, and introduction of an exogenous ER gene in cultured colon carcinoma cells resulted in marked growth suppression. Our data suggest that methylation associated inactivation of the ER gene in ageing colorectal mucosa could be one of the earliest events that predispose to sporadic colorectal tumorigenesis.

Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability.

Microsatellite instability has been observed in both sporadic and hereditary forms of colorectal cancer. In the hereditary form, this instability is generally due to germline mutations in mismatch repair (MMR) genes. However, only one in ten patients with sporadic tumours exhibiting microsatellite instability had a detectable germline mutation. Moreover, only three of seven sporadic tumour cell lines with microsatellite instability had mutations in a MMR gene, and these mutations could occur somatically. These results demonstrate that tumours can acquire somatic mutations that presumably do not directly affect cell growth but result only in genetic instability. They also suggest that many sporadic tumours with microsatellite instability have alterations in genes other than the four now known to participate in MMR.

Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers.

Chromosome deletions are the most common genetic events observed in cancer. These deletions are generally thought to reflect the existence of a tumour suppressor gene within the lost region. However, when the lost region does not precisely coincide with a hereditary cancer locus, identification of the putative tumour suppressor gene (target of the deletion) can be problematic. For example, previous studies have demonstrated that chromosome 18q is lost in over 60% of colorectal as well as in other cancers, but the lost region could not be precisely determined. Here we present a rigorous strategy for mapping and evaluating allelic deletions in sporadic tumours, and apply it to the evaluation of chromosome 18 in colorectal cancers. Using this approach, we define a minimally lost region (MLR) on chromosome 18q21, which contains at least two candidate tumour suppressor genes, DPC4 and DCC. The analysis further suggested genetic heterogeneity, with DPC4 the deletion target in up to a third of the cases and DCC or a neighbouring gene the target in the remaining tumours.

Mad-related genes in the human.

Resistance to the growth inhibitory effects of TGF-beta is common in human cancers. However, the mechanism(s) by which tumour cells become resistant to TGF-beta are generally unknown. We have identified five novel human genes related to a Drosophila gene called Mad which is thought to transduce signals from TGF-beta family members. One of these genes was found to be somatically mutated in two of eighteen colorectal cancers, and three of the other genes were located at chromosomal positions previously suspected to harbor tumour suppressor genes. These data suggest that this gene family may prove to be important in the suppression of neoplasia, imparting the growth inhibitory effects of TGF-beta-like ligands.

Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC.

Approximately 130,000 cases of colorectal cancer (CRC) are diagnosed in the United States each year, and about 15% of these have a hereditary component. Two well-defined syndromes, familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC), account for up to 5% of the total new cases of CRC. Truncating APC mutations are responsible for FAP, and defective mismatch repair genes cause HNPCC. However, the genes responsible for most of the familial cases are unknown. Here we report a mutation (T to A at APC nucleotide 3920) found in 6% of Ashkenazi Jews and about 28% of Ashkenazim with a family history of CRC. Rather than altering the function of the encoded protein, this mutation creates a small hypermutable region of the gene, indirectly causing cancer predisposition.

Crizotinib in patients with tumors harboring ALK or ROS1 rearrangements in the NCI-MATCH trial.

The NCI-MATCH was designed to characterize the efficacy of targeted therapies in histology-agnostic driver mutation-positive malignancies. Sub-protocols F and G were developed to evaluate the role of crizotinib in rare tumors that harbored either ALK or ROS1 rearrangements. Patients with malignancies that progressed following at least one prior systemic therapy were accrued to the NCI-MATCH for molecular profiling, and those with actionable ALK or ROS1 rearrangements were offered participation in sub-protocols F or G, respectively. There were five patients who enrolled on Arm F (ALK) and four patients on Arm G (ROS1). Few grade 3 or 4 toxicities were noted, including liver test abnormalities, and acute kidney injury. For sub-protocol F (ALK), the response rate was 50% (90% CI 9.8-90.2%) with one complete response among the 4 eligible patients. The median PFS was 3.8 months, and median OS was 4.3 months. For sub-protocol G (ROS1) the response rate was 25% (90% CI 1.3-75.1%). The median PFS was 4.3 months, and median OS 6.2 months. Data from 3 commercial vendors showed that the prevalence of ALK and ROS1 rearrangements in histologies other than non-small cell lung cancer and lymphoma was rare (0.1% and 0.4% respectively). We observed responses to crizotinib which met the primary endpoint for ALK fusions, albeit in a small number of patients. Despite the limited accrual, some of the patients with these oncogenic fusions can respond to crizotinib which may have a therapeutic role in this setting.

Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability.

Loss of imprinting (LOI) is an epigenetic alteration of some cancers involving loss of parental origin-specific expression of imprinted genes. We observed LOI of the insulin-like growth factor-II gene in twelve of twenty-seven informative colorectal cancer patients (44%), as well as in the matched normal colonic mucosa of the patients with LOI in their cancers, and in peripheral blood samples of four patients. Ten of eleven cancers (91%) with microsatellite instability showed LOI, compared with only two of sixteen tumors (12%) without microsatellite instability (P < 0.001). Control patients without cancer showed LOI in colonic mucosa of only two of sixteen cases (12%, P < 0.001) and two of fifteen blood samples (13%, P < 0.001). These data suggest that LOI in tumor and normal tissue identifies most colorectal cancer patients with microsatellite instability in their tumors, and that LO! may identify an important subset of the population with cancer or at risk of developing cancer.

Genetic instability occurs in the majority of young patients with colorectal cancer.

Replication errors (RER) associated with genetic instability have been found in cancers of several different types and particularly in the tumours of patients with hereditary non-polyposis colorectal cancer (HNPCC). We have here determined the prevalence of such instability in relation to age among patients without HNPCC. Colorectal cancers (CRCs) in the majority of patients 35 years of age or younger exhibited instability (58% of 31 patients), whereas CRCs from patients older than 35 uncommonly did (12% of 158, p < 0.0001). Twelve of the patients under 35 with instability were evaluated for alterations of mismatch repair genes, and five were found to harbour germline mutations. These data suggest that the mechanisms underlying tumour development in young CRC patients differ from those in most older patients, regardless of HNPCC status. The results have important implications for genetic testing and management of young CRC patients and their families.

Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disorder characterized by the early onset of colorectal cancer and linked to germline defects in at least four mismatch repair genes. Although much has been learned about the molecular pathogenesis of this disease, questions related to effective presymptomatic diagnosis are largely unanswered because of its genetic complexity. In this study, we evaluated tumors from 74 HNPCC kindreds for genomic instability characteristic of a mismatch repair deficiency and found such instability in 92% of the kindreds. The entire coding regions of the five known human mismatch repair genes were evaluated in 48 kindreds with instability, and mutations were identified in 70%. This study demonstrates that a combination of techniques can be used to genetically diagnose tumor susceptibility in the majority of HNPCC kindreds and lays the foundation for genetic testing of this relatively common disease.

Clonal analysis of human colorectal tumors.

The clonal composition of human colorectal tumors was studied by means of restriction fragment length polymorphisms (RFLPs). First, X-linked RFLPs were used to examine the pattern of X chromosome inactivation in colorectal tumors of females. All 50 tumors examined showed monoclonal patterns of X chromosome inactivation; these tumors included 20 carcinomas as well as 30 adenomas of either familial or spontaneous type. Second, RFLPs of autosomes were used as clonal markers to detect the somatic loss or gain of specific chromosomal sequences in colorectal tumors. Among other changes, it was found that somatic loss of chromosome 17p sequences occurred in over 75 percent of the carcinomas examined, but such loss was rare in adenomas. These data support a monoclonal origin for colorectal neoplasms, and suggest that a gene on the short arm of chromosome 17 may be associated with progression from the benign to the malignant state.

Use of restriction fragment length polymorphisms to determine the clonal origin of human tumors.

A novel strategy to determine the clonal origin of human tumors has been devised. The strategy involves the use of a cloned polymorphic X-chromosomal gene and two restriction endonucleases. The first endonuclease distinguishes the paternal and maternal copies of the gene through a DNA polymorphism of restriction fragment length. The second endonuclease distinguishes active from inactive copies of this gene through changes in DNA methylation. As illustrations of this strategy, three human cancers were each shown to be monoclonal. The analysis described should have a wide variety of clinical and experimental applications.

Hypomethylation of DNA from benign and malignant human colon neoplasms.

The methylation state of DNA from human colon tissue displaying neoplastic growth was determined by means of restriction endonuclease analysis. When compared to DNA from adjacent normal tissue, DNA from both benign colon polyps and malignant carcinomas was substantially hypomethylated. With the use of probes for growth hormone, gamma-globin, alpha-chorionic gonadotropin, and gamma-crystallin, methylation changes were detected in all 23 neoplastic growths examined. Benign polyps were hypomethylated to a degree similar to that in malignant tissue. These results indicate that hypomethylation is a consistent biochemical characteristic of human colonic tumors and is an alteration in the DNA that precedes malignancy.

Gene amplification of c-myc and N-myc in small cell carcinoma of the lung.

The relationship of the copy numbers of the c-myc and N-myc oncogenes to tumor formation and progression was studied in small cell carcinoma of the lung. When 96 neoplastic lesions from 45 patients were examined, these lesions could be grouped into three categories: high copy (tumors with greater than 3 copies of the N-myc or c-myc gene per haploid genome), middle copy (1.5 to 3 copies per genome), and normal copy. Fourteen of the patients had middle copy tumors, but this was almost always a result of chromosome duplication rather than the amplification of a small genetic locus. In contrast, five patients had high copy tumors, with the increased copy number in each case due to gene amplification. The amplification did not occur in a heterogeneous fashion within individual patients, since all metastatic lesions from patients with high copy lung tumors were also high copy, while none of 41 metastatic lesions from the other patients were high copy. These data suggest that gene amplification is an important step in neoplastic growth in a subset of patients with small cell carcinoma of the lung and that this genetic event occurs relatively early (before metastasis) in this subset.

Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors.

Colorectal (CR) tumors are usually curable if detected before metastasis. Because genetic alterations are associated with the development of these tumors, mutant genes may be found in the stool of individuals with CR neoplasms. The stools of nine patients whose tumors contained mutations of K-ras were analyzed. In eight of the nine cases, the ras mutations were detectable in DNA purified from the stool. These patients included those with benign and malignant neoplasms from proximal and distal colonic epithelium. Thus, colorectal tumors can be detected by a noninvasive method based on the molecular pathogenesis of the disease.

Allelotype of colorectal carcinomas.

To examine the extent and variation of allelic loss in a common adult tumor, polymorphic DNA markers were studied from every nonacrocentric autosomal arm in 56 paired colorectal carcinoma and adjacent normal colonic mucosa specimens. This analysis was termed an allelotype, in analogy with a karyotype. Three major conclusions were drawn from this analysis: (i) Allelic deletions were remarkably common; one of the alleles of each polymorphic marker tested was lost in at least some tumors, and some tumors lost more than half of their parental alleles. (ii) In addition to allelic deletions, new DNA fragments not present in normal tissue were identified in five carcinomas; these new fragments contained repeated sequences of the variable number of tandem repeat type. (iii) Patients with more than the median percentage of allelic deletions had a considerably worse prognosis than did the other patients, although the size and stage of the primary tumors were very similar in the two groups. In addition to its implications concerning the genetic events underlying tumorigenesis, tumor allelotype may provide a molecular tool for improved estimation of prognosis in patients with colorectal cancer.

Multiclonal origin of polyps in Gardner syndrome.

Electrophoretic analysis of glucose-6-phosphate dehydrogenase was performed on polyp tissue from three black female patients with Gardner syndrome and who are heterozygous for the A and B forms of this enzyme. Polyp tissues from the three patients displayed the AB phenotype. This finding suggests a multiclonal origin of polyps in Gardner syndrome. Studies of tumors originating from such polyps may provide information about the sequence of cellular events leading to malignant transformation.

Clues to the pathogenesis of familial colorectal cancer.

A predisposition to colorectal cancer is shown to be linked to markers on chromosome 2 in some families. Molecular features of "familial" cancers were compared with those of sporadic colon cancers. Neither the familial nor sporadic cancers showed loss of heterozygosity for chromosome 2 markers, and the incidence of mutations in KRAS, P53, and APC was similar in the two groups of tumors. Most of the familial cancers, however, had widespread alterations in short repeated DNA sequences, suggesting that numerous replication errors had occurred during tumor development. Thirteen percent of sporadic cancers had identical abnormalities and these cancers shared biologic properties with the familial cases. These data suggest a mechanism for familial tumorigenesis different from that mediated by classic tumor suppressor genes.

Identification of p53 gene mutations in bladder cancers and urine samples.

Although bladder cancers are very common, little is known about their molecular pathogenesis. In this study, invasive bladder cancers were evaluated for the presence of gene mutations in the p53 suppressor gene. Of 18 tumors evaluated, 11 (61 percent) were found to have genetic alterations of p53. The alterations included ten point mutations resulting in single amino acid substitutions, and one 24-base pair deletion. In all but one case, the mutations were associated with chromosome 17p allelic deletions, leaving the cells with only mutant forms of the p53 gene products. Through the use of the polymerase chain reaction and oligomer-specific hybridization, p53 mutations were identified in 1 to 7 percent of the cells within the urine sediment of each of three patients tested. The p53 mutations are the first genetic alterations demonstrated to occur in a high proportion of primary invasive bladder cancers. Detection of such mutations ex vivo has clinical implications for monitoring individuals whose tumor cells are shed extracorporeally.